dendritic cell protocols

Dendritic cells isolated from lymphnodes represent “interdigitating” DCs that are localized in T-dependent regions of lymph nodes.. After a 25-min incubation on ice, wash cells twice and

Humana Press

Dendritic Cell Protocols

Edited by

Dendritic Cell Protocols

Edited by

3

From: Methods in Molecular Medicine, vol 64: Dendritic Cell Protocols

Edited by: S P Robinson and A J Stagg © 2001 Humana Press Inc., Totowa, NJ

Isolation of Dendritic Cells from Mouse Lymph NodesDmitry Gabrilovich

1 Introduction

Lymph nodes are the primary sites of T-cell stimulation by dendritic cells(DC) After contact with antigens, DCs migrate to draining lymph nodes from

the skin and other tissues (1–3) Investigation of the morphology and function

of lymph node DCs may provide important information about the role of thesecells in normal and pathological conditions Therefore, lymph nodes are popu-lar sites for the isolation of dendritic cells Dendritic cells isolated from lymphnodes represent “interdigitating” DCs that are localized in T-dependent regions

of lymph nodes DCs represent about 1% of the total population of lymph nodecells Therefore, in order to perform almost any functional tests, the DC fractionshould be enriched The most practical way to enrich the DC fraction is to use

a density gradient Several gradients—metrizamide (4), Nycodenz (5), and Percoll (6)—have successfully been used for enrichment of DCs obtained from

different sources When isolating DC from lymph nodes, density gradient ration produces a population of DC with a purity of 40–50% Most contaminat-ing cells are lymphocytes with a small fraction (usually less than 5%) ofmacrophages The choice of lymph nodes is dependent on the purpose of theexperiment The most commonly used lymph nodes are axillary, inguinal, andpopliteal DCs can be further enriched using monoclonal antibodies and flowcytometric cell sorting, magnetic beads separation, panning, or cytotoxic elimi-nation with complement All these methods are based on the negative selection

sepa-of DCs using anti-T, anti-B, and anti-macrophage antibodies Since the firststep of isolation involves gradient centrifugation, granulocyte contamination

is negligible and further purification steps do not require use of cyte antibodies

anti-granulo-2 Materials

1 Sterile dissecting forceps and scissors for lymph node extraction

2 Sterile 6-well plates, 50-mL conical tubes, and 15-mL conical tubes (Falcon,Becton Dickinson, Franklin Lakes, NJ) Sterile 70-µm cell strainers (Falcon), 5 mLsyringes, and 5 mL and 10 mL pipets

3 Fetal calf serum (FCS) (HyClone, Logan, UT) Culture media DMEM and RPMI

1640 (Gibco-BRL, Grand Island, NY) supplemented with antibiotics We monly use a combination of penicillin, streptomycin, and Fungizone (antibiotic–antimycotic, Gibco-BRL) DMEM can be used without serum RPMI 1640 should

com-be supplemented with 10% FCS (RPMI-FCS)

4 Metrizamide gradient Dissolve 7.25 g metrizamide with 45 mL RPMI in a 50-mLtube It usually takes 15–20 min Sterilize the gradient by passing through a 0.45µmfilter Add 5 mL of FCS, mix and prepare 2.5 mL aliquots Store the gradient at–30°C We use metrizamide produced by Nygaard, Norway Metrizamide is alsoproduced by Sigma, and we have had satisfactory results with Sigma’smetrizamide

5 Hemocytometer, and microscope with 400× magnification

6 PE conjugated anti-B7-2 (CD86) antibody, FITC conjugated anti-CD11c (N418)antibody, and PE and FITC conjugated mouse IgG2a and IgG2b as isotype con-trol (Pharmingen)

3 Methods

3.1 Isolation of DC by Density Gradient Centrifugation

1 Place a cell strainer into one well of a 6-well plate Fill the well with DMEM.Prepare as many wells as necessary

2 Sacrifice mice using one of the methods approved by the appropriate institutionalreview board Extract the lymph nodes from at least three mice and put the lymphnodes together on the cell strainer submerged in DMEM Make sure that thelymph nodes are covered with the medium

3 Remove the plunger from a syringe and use it to press the lymph nodes throughthe mesh of the cell strainer Make sure that lymph nodes are completely shat-tered on the mesh Finally, wash the strainer with 3–4 mL of DMEM

4 Discard cell strainers and collect cells from the well into a 15 mL conical tube

Wash cells once with DMEM by centrifuging for 5 min at 300g Resuspend cells

iden-morphological or functional studies (see Notes 1 and 2) To confirm the purity of the samples, cells can be analyzed by flow cytometry (see Subheading 3.3.).

anti-MO Add 3 mL of each antibody at a concentration of 1 mg/mL in buffered saline (PBS) to separate wells

phosphate-2 After at least 60-min incubation at room temperature, remove the antibody

solu-tion and wash the wells four to five times with PBS (see Note 4).

3 Wash the DC fraction derived by density gradient centrifugation (see

Subhead-ing 3.1.) once in PBS and resuspend in 100µL of hybridoma supernatants ofanti-CD4 antibody (L3T4, TIB-207, ATCC, Rockville, MD), 100 µL of hybri-doma supernatants of anti-CD8 antibody (Lyt-2.2, TIB-210, ATCC), and 20µL

of anti-F4/80 antibody (Serotec, Raleigh, NC) (see Note 5).

4 After a 25-min incubation on ice, wash cells twice and resuspend in 3 mL of PBScontaining 0.1% mouse serum

5 Transfer the cell suspension to a well coated with anti-rat immunoglobulin bate cells on the plates for 60–90 min at 4°C

Incu-6 Harvest the nonadherent DC enriched fraction using a 5 mL pipete Gently washthe well with ice cold PBS to remove any partially adherent cells and add these to

the nonadherent fraction (see Note 5).

7 Concentrate the harvested cells by centrifuging at 300g Resuspend the cell

pel-let in 3 mL of cold PBS with 0.1% serum and transfer into a well coated withanti-mouse immunoglobulin Incubate for another 60-90 min and then collectcells as described previously Cells can now be resuspended in RPMI-FCS and

used for further study (see Note 5).

3.3 Analysis of DC Purity by Flow Cytometry

We routinely use double labeling with CD11c (N418) antibody and B7-2 (CD86) antibody to identify DC as CD11c+ CD86+ cells on the flowcytometer

anti-1 Transfer 100µL of the purified DC cell suspension into two tubes for flowcytometry labeled “test” and “control.”

2 Wash cells once with PBS and resuspend in 100µL of PBS

3 Add 5 µL of anti-B7-2 and anti-CD11c antibodies into the “test” tube and 5 µL ofisotype control antibodies into the “control” tube

4 Incubate on ice for 25 min, then wash twice with 2 mL of cold PBS and analyze

on the flow cytometer (see Note 6).

4 Notes

1 Generally, one can expect to isolate around 5–10×103DCs from one lymph node Forfunctional tests, we usually collect lymph nodes from three or four mice, which pro-vides a sufficient number of cells (around 2×105) for several functional tests

2 It is recommended that 10–5M of `-mercaptoethanol is added to the culturemedium while growing mouse cells However, `-mercaptoethanol should beadded after completion of all isolation procedures, since even in low concentra-tion it may affect the binding of antibodies to the cells

3 Owing to the relatively low number of cells, lymph nodes are not the best source

of highly purified DCs However, if the experimental goal makes it necessary touse highly purified lymph node DCs, we suggest using the panning technique asopposed to complement-dependent cytotoxicity The latter method usually results

in the nonspecific loss of some DC If the investigator has access to a flow cellsorter, this method, as well as the magnetic bead separation technique, may pro-vide good alternatives For all sorting procedures, the author recommends using

at least six mice per sample

4 Panning plates may be stored overnight at 4°C Collected antibody solution can

be used again several times

5 Anti-Thy-1.2 antibody produced by hybridoma supernatant (ATTC TIB 107) can

be used instead of anti-CD4 and anti-CD8 antibodies All these antibodies are ratIg2b In the first panning step, T cells and macrophages are eliminated using anti-rat monoclonal antibodies In the second step, B cells are removed using anti-mouse immunoglobulin antibody

6 It is important to perform all procedures at 4°C, because DCs readily adhere toplastic and some cells may be lost if the incubation is performed at room tem-perature The purity of DCs can be verified using anti-CD11c and B7-2 antibod-ies The final purity of the DC fraction is usually above 95% following panning.One can expect to obtain at least 105 highly purified DCs from five or six mice

7 A typical example of double labeling of lymph node DCs with CD11c and B7-2

antibodies is shown Fig 1 Cells were isolated and labeled with antibodies as

described in “Methods.” Analysis was performed using FACSCalibur flowcytometer (Becton Dickinson, Mountain View, CA) It is important to note thatthere is no single marker that would allow for detection of 100% of lymph nodeDCs N418 (CD11c) may bind to some macrophages, whereas B7-2 binds prima-rily to only mature DCs The investigator may choose to use a combination ofother DC markers depending on the goal of the study

References

1 Steinman, R M (1991) The dendritic cell system and its role in immunogenicity

Annu Rev Immunol 9, 271–296.

2 Knight, S C., and Stagg, A J (1993) Antigen-presenting cell types Curr Opin.

Immunol 5, 374–382.

3 Hart, D N J (1997) Dendritic cells: Unique leukocyte populations which control

the primary immune response Blood 90, 3245–3287.

4 Knight, S C., Farrant, J., Bryant, A., et al (1986) Non-adherent, low density cellsfrom human peripheral bloodcontain dendritic cells and monocytes, both with

veiled morphology Immunology 57, 595–598.

5 McLellan, A D., Starling,G C., and Hart, D N C (1995) Isolation of human

blood dendritic cells by Nycodenz discontinuous gradient centrifugation J.

Immunol Methods 184, 81–85.

6 Young, J W and Steinman, R M (1988) Accessory cell requirements for theMLR and polyclonal mitogens, as studied with a new technique for enriching

blood dendritic cells Cell Immunol 111, 167–171.

Fig 1 Flow cytometry demonstrates the purity of DC isolated from lymph nodesfollowing density gradient centrifugation

9

From: Methods in Molecular Medicine, vol 64: Dendritic Cell Protocols

Edited by: S P Robinson and A J Stagg © 2001 Humana Press Inc., Totowa, NJ

Isolation of Mouse Spleen Dendritic Cells

Andrew J Stagg, Fiona Burke, Suzanne Hill, and Stella C Knight

1 Introduction

It is now over 20 years since dendritic cells (DC) were first identified in and

isolated from the spleens of mice (1,2) and they continue to be a much-studied

population Only a small proportion of spleen cells are DC, but the large size ofthe organ means that useful numbers of DC can still be purified In recent yearsthe ability to grow cells with the phenotypic and functional properties of DCfrom bone marrow progenitors has opened new avenues of research However,the relationship of cells grown in this way to DC populations in vivo isunknown and the need remains to study DC present in tissues

Spleen DC are heterogeneous with differences in phenotype, function, and

microanatomical location (3,4) At least two major subsets are recognized, and

these can be discriminated on the basis of the presence or absence of a surface homodimer of the CD8 molecule The freshly isolated CD8 +population is DEC-205+, CD24+, CD11b-, 33D1-, CD4-, whereas the CD8 -subset is DEC205-, CD24-, CD11b+, 33D1+, CD4- Both subsets expressCD11c, and this marker appears to be expressed selectively on DC and in themouse can be used as a pan-DC marker The CD8 +population predomi-nately localizes in the T-cell areas of the white pulp and corresponds to inter-digitating cells In the steady state, the CD8 -population is probably localizedpredominately in the marginal zone, between the red and white pulp, but mobi-lizes into the T-cell areas in response to lipopolysaccharide (LPS) administra-

cell-tion (5) This marginal zone DC populacell-tion has a higher phagocytic activity and turnover rate than the interdigitaing cells (6) The CD8 +and CD8 -populations may be cells of lymphoid and myeloid lineages, respectively They

can both activate resting T cells, but may stimulate different types of responses.The CD8 + population has been reported to drive preferentially Th1responses, whereas presentation of antigen by the CD8 -may favor Th2

responses (7) CD8 +DC can also kill activated T cells via Fas-mediated

apoptosis (8) The division of spleen DC into “lymphoid” and “myeloid”

popu-lations is probably an oversimplification, and recent evidence suggests furtherheterogeneity with the description of a third, CD4+, spleen DC subset (9).

There are many published protocols for isolating mouse spleen cells and inchoosing among these methods two factors should be borne in mind First,different methods may favor the recovery of particular DC subsets at theexpense of others This can be a problem if the intention is to recover a repre-sentative sample of the total spleen DC, but it can also be turned to theinvestigator’s advantage in the purification of particular subsets Second, DCmay be altered phenotypically or functionally by the isolation process itself.This modulation occurs in methods in which DC are cultured for prolongedperiods, because in vitro culture is sufficient to induce DC maturation Changes

in properties of DC may also occur during positive selection with monoclonalantibodies or digestion of tissue with proteolytic enzymes For instance, colla-genase preparations are likely to contain significant concentrations of endot-oxin that may affect DC

In this chapter we describe a basic method for the enrichment of mousespleen DC that involves overnight culture and separation on hypertonicmetrizamide gradients and provide a suggested protocol for the further purifi-cation of these cells We also describe an alternative method for spleen DC thatavoids the need for culture and discuss how the choice of method for initialpreparation of a spleen cell suspension can be used to influence the recovery ofparticular DC subsets

2 Materials

1 Specific pathogen free mice The commonly used strains in our laboratory areBALB/c, CBA, and C3H We have used mice of either sex, and they are usuallyaged 6–12 wk

2 Dissecting board or paper tissues

3 70% ethanol

4 Sterile surgical instruments (forceps and scissors)

5 Complete medium: Dutch modification of RPMI-1640 (Sigma; cat no.R-7638)

supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM

L-gluta-mine,100U/mL penicillin/streptomycin, and 5×10–5M 2-mercaptoethanol (2-ME)

(see Note 1).

6 HEPES-buffered RPMI-1640 (Sigma, cat no R-5886)

7 Metal cell strainers

8 60 mm Petri dishes (Nunc or Sterilin)

9 2 mL and 1 mL syringes (Terumo).

10 10 mL conical-bottomed tubes (Sterilin; cat no 144AS)

11 Disposable Pasteur pipets (Alpha Labs; cat no LW4005) or sterilized glassequivalents

12 Small filters for sterilization (GelmanSciences; cat no 6224184 [0.45mm] orcat no 6224192 [0.22 µm])

13 Collagenase digestion mix: 1mg/mL collagenase D (Roche Molecular Products;cat no 1088 866), 20 µg/mL DNase I (Roche Molecular Products; cat no 1284

982), 2% FCS in HEPES-buffered RPMI-1640 (see Notes 2 and 3).

To prepare collagenase stock:

a Dissolve 500 mg of collagenase D in 50 mL serum-free HEPES bufferedRPMI-1640 (10 mg/mL)

b Filter sterilize (0.45 µm)

c Store in aliquots at –20°C

d Avoid repeated freezing and thawing

e Thaw aliquots as required and keep on ice until used

To prepare DNase I stock:

a Dissolve 100 mg in 10 mL dH20 (10 mg/mL)

b Filter sterilize

c Store in aliquots at –20°C

d Avoid repeated freezing and thawing

To make 10 mL of digestion mix combine:

a 1 mL collagenase D stock

b 20 µL DNase I stock

c 0.2 mL FCS

d 8.8ml HEPES buffered RPMI-1640

Keep on ice until use

14 26G × 1/2 in needles

15 Disposable scalpels or scalpel blades

16 T25 tissue culture flasks (Falcon; cat no.353014) (see Note 4).

17 Cell scrapers (Falcon; cat no 3085)

18 Analytical grade metrizamide (Nycomed; cat no.22.20.10) (see Note 5).

19 Sterile 5 mL (75 mm × 12 mm) push cap round bottomed tubes (Sarstedt; cat no.55.476.013)

20 MiniMACS buffer: PBS containing 5% bovine serum albumin (BSA) and 5 mM

EDTA Filter sterilize Handle carefully to avoid frothing (see Note 6).

21 Heat-inactivated normal mouse serum

22 Monoclonal antibodies and immunomagnetic microbeads (see Note 7) These include:

a “Fc-Block” (PharMingen; cat no 01241A/D) (see Note 8).

b Microbeads coated with anti-CD11c (N418) (Miltenyi; cat no.520-01)

c Anti-CD11c-FITC (clone HL3) (PharMingen; cat no.09704A/D) For someapplications the same antibody labeled with an alternative fluorochrome (e.g.,phycoerythrin) may also be required

d Anti-CD45R-FITC (B220) (PharMingen; cat no.01124A/D).

e Microbeads coated with anti-FITC (Miltenyi; cat no.487-01)

23 MiniMACS magnet and holder or the varioMACS system (Miltenyi) (see Note 9)

24 MiniMACS columns (Type MS+/RS+for miniMACS or varioMACS and/or Type

LS+/VS+ for varioMACS, Miltenyi) (see Note 9).

3 Methods

3.1 Preparation of Single Cell Suspensions from Spleens

This section describes removal of the mouse spleen and presents three ferent methods for producing a single-cell suspension from the organ The way

dif-in which the choice of methods dif-influences the recovery of DC is discussed.3.1.1 Removal of the Spleen

1 Kill the mouse by cervical dislocation

2 Lay mouse on dissecting board, “left side” uppermost

3 Surface-sterilize the skin using 70% ethanol or a proprietary compound

4 Using one set of sterile surgical instruments (forceps and scissors), cut throughthe skin just below the ribcage and visualize the spleen

5 Using a second, smaller, set of instruments, remove the spleen, trimming awayany fatty tissue

6 Place spleen into complete medium at room temperature (see Note 10) Spleens

from multiple animals can be pooled

3.1.2 Preparation of a Single-Cell Suspension using a Metal SieveThis has been our routine method for many years It avoids the use of pro-teolytic enzymes, gives good recovery of DC numbers, and, in conjunctionwith overnight culture and metrizamide separation, yields a mixture of CD8 +and CD8 - DC (see Fig 1).

1 Strain spleens by pouring through a sterile metal cell-strainer Discard medium

2 Place the strainer containing spleens into a 60 mm Petri dish and add a few liters of fresh medium

milli-3 Using the barrel from a 2 mL syringe, press the spleens through the strainer.Continue until only a little fibrous tissue remains in the strainer

4 Remove the strainer and place in upturned lid of the Petri dish

5 Reinsert plunger into syringe barrel and use to transfer spleen cell suspension to

a 10 mL conical tube (A larger tube or replicate tubes will be required for tiple spleens.)

mul-6 Using fresh medium and a Pasteur pipet rinse the Petri dish and the cell strainer

to ensure that all cells have been recovered Pool with the rest of the spleen cellsuspension

7 Top-up tube with complete medium to appropriate volume (see below).

3.1.3 Preparation of a Single-Cell Suspension using CollagenaseThis method gives an overall increase in DC yield and improves the recov-ery of the tightly tissue-bound CD8 + DC (Fig 1) The use of proteolytic

enzymes may be undesirable for some applications

1 Place 5 mL of digestion mix into a Petri dish

2 Put a small needle (26G × 1/2 in is ideal) on a 1 mL syringe and fill with tion mix

diges-3 Gently inject the first spleen with 0.5–1 mL of digestion mix Initially insert theneedle just inside the spleen at the narrowest part of the capsule Inject approx

100µL, advance the needle slightly, and then inject again Continue in this ion This may take some practice The spleen will distend and change from a darkmaroon color to a reddish orange

fash-4 Using the needle tear open the spleen in a second (empty) Petri dish

5 Place the spleen back in the first Petri dish containing the digestion mix

6 Transfer the released cells in the second dish to a conical tube and place on ice.Rinse this dish with more digestion mix and pool with the other cells on ice

Fig 1 Method used to prepare a singlecell suspension from spleens influences the number (A) and type (B) of DC obtained Spleen cells were obtained by the strainer,

collagenase or manual extraction techniques as described in the text The absolutenumber of CD11c+DC obtained following overnight culture and separation onmetrizamide was determined by flow cytometry with simultaneous acquisition of FlowCount fluorospheres (Beckman Coulter) The proportion of CD8+DC was determined

by double staining with anti-CD8_

7 Repeat steps 1–6 with the next spleen, pooling released cells and spleens.

8 Using a disposable scalpel or scalpel blade cut up spleens into small fragments

9 Transfer to a T25 tissue culture flask and incubate with gentle shaking at 37°C

for approx 60 min (see Note 11).

10 At the end of the incubation, collect contents of dish Rinse dish with a smallvolume of digestion mix and pool together

11 Press contents through a cell strainer as described previously

12 Pool cells with those already on ice Rinse dish in digestion mix or medium andagain pool with the other cells

13 Spin down (350g, 5 min), discard supernatant and gently resuspend cell pellet in

10 mL complete medium

14 Spin down again and resuspend in complete medium Adjust to required volume

(see below).

3.1.4 Preparation of a Single-Cell Suspension

by the “Manual Extraction” Method

This very gentle method yields DC that are almost exclusively CD8 - (Fig 1),

so this approach may be useful as an early step in purifying this subset

1 Place some complete medium in a Petri dish and place the dish at an angle byresting it partially on its lid

2 Using forceps, make a hole in the capsule at one end of the spleen

3 Place the spleen on the sloping Petri dish, punctured end facing “down the hill.”Hold in place with forceps

4 Using a cell scraper gently press cells out of the spleen by working the scraperfrom the middle to the lower end of the spleen

5 When capsule is clear turn the spleen round, make a hole at the other end of it andwork the rest of the cells out

6 When all cells have been removed, discard the empty capsule and transfer thecells to a conical tube

7 Repeat the procedure with additional spleens, pooling the released cells

8 Adjust to the appropriate volume (see below).

3.2 Enrichment of DC Using Metrizamide Gradients

Following overnight culture, a single separation step on a metrizamide dient enriches DC up to 100-fold The separation is based partly on density andpartly on differential shrinkage of cells following exposure to the metrizamide,which is slightly hypertonic Following centrifugation, low density cells (LDC)that stay up on the gradient contain most of the DC, whereas the lymphocyte-rich high density cells form a pellet

gra-3.2.1 Preparation of Metrizamide Gradients

1 Weigh out 7.25 g of metrizamide and place in 50 mL conical tube

2 Add a total of 45 mL HEPES-buffered RPMI-1640 To get the metrizamide intosolution, it is easier to add the medium in several stages (perhaps 15 mL at a

time), mix gently, and then leave to stand for a while after each addition Avoidinverting the tube as metrizamide is quite sticky and can be “lost” stuck to the top

of the tube

3 Add 5 mL of FCS Adding this separately makes the metrizamide easier to

dis-solve Note that the total volume of medium added is 50 mL so the final solution

is less than 14.5% w/v (actually 13.7% w/v)

4 Filter sterilize (0.45 µm), divide into 2 mL aliquots and store at –20°C (see Note 13).

5 On the day of taking spleens calculate the required number of metrizamide

aliquots (1–2 per spleen–see below) and place in a fridge overnight to defrost.

3.2.2 Overnight Culture of Spleen Cells

Overnight culture of spleen cells prior to centrifugation on metrizamideprobably contributes to DC separation in three ways First, “maturation” of the

DC in culture increases their tendency to stay up on the gradient In line withthis, mature DC can be separated from lymph nodes without the need for prior

culture of the cells (see Chapter 1), presumably because the lymph node DC

population is more mature in vivo Second, culture allows migration from

tis-sue fragments of tightly bound DC (see Note 14) Third, some contaminating

cells with prolonged adherence properties (e.g., macrophages) may be removed

by culture

1 Prepare single-cell suspensions from spleens by one of the methods describedabove

2 Resuspend in 5-10 mL of complete medium per spleen (see Note 15).

3 Add the spleen cell suspension to T25 tissue culture flasks, putting 5ml into each

flask (see Note 15).

4 Culture overnight at 37°C in a humidified incubator containing 5% CO2in air

(see Note 16).

3.2.3 Separation on Metrizamide

1 Remove the thawed metrizamide from the fridge and allow it to come to room

temperature while preparing the spleen cells (see Note 17).

2 Take flasks containing the overnight cultures of spleen cells from the incubator

3 Resuspend the cells and dislodge loosely adherent populations by pipeting themedium containing the cells up and down with a Pasteur pipet This can be donefairly vigorously

4 Transfer each 2 mL aliquot of metrizamide to a 10 mL conical centrifuge tube

5 Carefully overlay each metrizamide gradient with 5 mL of cell suspension (0.5–1spleen per gradient) This can be done with a Pasteur or a syringe and filling tubeaccording to preference but does require a little practice Monitor the gradient asthe cells are added to ensure that a “clean” interface forms between themetrizamide and the cell suspension Mixing of the two will adversely effect theseparation

6 Centrifuge at room temperature at 650g for 10 min (No brake!)

7 Using a Pasteur pipet, recover the LDC from the interface between themetrizamide and medium Avoid taking up any of the pellet cells or any fattymaterial that may float at the top of the medium

8 If desired, the high density cell pellet can also be recovered (see Note 18).

9 Centrifuge the LDC suspension (650g, 10 min, room temperature).

10 Discard the supernatant and gently resuspend the pellet in complete medium(approx 5 mL per tube)

11 Spin down again This step can be more gentle than previous centrifugations

(350g for 5 min) as the majority of metrizamide has now been removed.

12 Discard the supernatant and resuspend the pellet in a small volume of medium(usually 1–2 mL depending on the number of spleens) pooling the contents ofreplicate tubes as appropriate

13 Perform a cell count Expect to recover in the order of 5×105to 1×106LDC perspleen depending on factors such as age and strain of mouse and the cleanliness

of the animal facility (see Note 19).

3.3 Further Purification of DC by Immunomagnetic Separation

The LDC preparation obtained by centrifugation over metrizamide will erally contain 40–60% CD11c+ DC (see Note 20) Some workers report up to

gen-80% purity Almost all of the contaminating cells are a population of B cells([CD19+CD45R(B220)+CD11c-]) These are larger cells than the majority ofthe spleen B-cell population (which is presumably why they separate in theLDC fraction) and may correspond to marginal zone B cells It is straightfor-ward to purify further the DC by positive selection using CD11c or by deplet-ing the CD45R+population (Fig 2) DC prepared by both approaches stimulate

an allogeneic mixed leukocyte reaction (Fig 3); the CD45R+cells stimulatemore weakly

3.3.1 Positive Selection of CD11c+ DC

1 In a 5 mL push-cap tube, wash LDC into cold MiniMACS buffer by diluting the

cells into the buffer and pelleting by centrifugation (350g) In a typical

experi-ment we might use 6×106 LDC

2 Discard the supernatant and gently resuspend the cells in the residual volume(approx 100 µL) (see Note 21).

3 On ice add the following:

15µl heat inactivated normal mouse serum

1.5µL “Fc-Block”

20µL immunomagnetic microbeads coated with anti-CD11c

4 Transfer to a fridge and incubate for 15 min (see Note 22)

5 Wash cells by topping-up the tube with cold miniMACS buffer and pelleting the

cells by centrifugation in the cold (400g).

6 Discard the supernatant and repeat washing step

7 Gently resuspend the cells in the residual volume (approx 100 µL).

8 Meanwhile prepare the miniMACS columns (Type MS):

a Assemble the column into the miniMACS magnet and place on holder

(see Note 9) Do not use a flow restrictor (i.e., set up for positive selection).

b Adding 500 µL of miniMACS buffer to the top of the tube and allow to washthrough the column The washing fluid (which will be turbid) can be col-lected and discarded

9 Add the cells to the top of the column and allow them to enter it

10 Add 500 µL to of miniMACS buffer to the top of the column and allow it to washthrough If desired, these washings can be collected from the bottom of the col-umn as a negative fraction

11 Wash the column twice by passing 500 µL of miniMACS buffer through it oneach occasion

12 Remove column from the magnet and, at a distance well removed from the

mag-Fig 2 DC can be further purified from the LDC preparation by either positive or

negative selection using antibodies to CD11c or CD45R(B220), respectively DCdepleted populations are obtained in parallel

netic field, add 1 mL of miniMACS buffer to the top of the tube and press thoughthe column using the plunger supplied.

13 Collect the positively selected cells from the bottom of the column

14 If desired, aliquots of cells can be removed at steps 7, 10, and 13 and labeled with

FITC conjugated anti-CD11c (clone HL3) (1.5 µL, 20 min on ice) in order tomonitor the purification process

15 The postively selected DC can be washed into complete medium (see Note 23) or

pelleted and passed over a second column A second pass can improve purity but at

a cost of additional cell losses Expect a purity of 95% (Fig 2).

3.3.2 Depletion of CD45R + Cells.

This is an alternative approach when there are concerns about coating theselected DC population with anti-CD11c We have generally used an indirectapproach in which cells are labeled with FITC-conjugated anti-CD45R andthen with anti-FITC immunomagnetic beads However, beads coated with anti-CD45R are also available, and these could be used in a direct labeling approach

1 Wash LDC into miniMACS buffer as in Subheading 3.3.1.

2 On ice add the following:

15µL heat inactivated normal mouse serum

Fig 3 DC, enriched by either positive or negative immunomagnetic selection,

stimulate a Primary mixed leukocyte reaction (MLR) Unseparated LDC and DCenriched or depleted preparations were irradiated (2000r) and used to stimulate 25000allogeneic lymph node cells in a 20µL hanging drop culture system Proliferation wasassessed by 3H-thymidine incorporation on day four of culture

1.5µL “Fc-Block.”

5µL anti-CD45R-FITC

3 Incubate in fridge for 15 min

4 Wash twice as described in Subheading 3.3.1.

5 Add 10 µL of anti-FITC coated immunomagnetic microbeads

6 Incubate in fridge for 15 min

7 Wash twice

8 Meanwhile prepare the miniMACS column (Type MS) The procedure is as

de-scribed in Subheading 3.3.1., except the flow restrictor supplied with the

col-umn is fitted prior to the initial washing of the colcol-umn

9 Add the cells, in approx 100 µL of miniMACS buffer to the top of the columnand let them run in

10 Add 500 µL of buffer to the top of the column and collect the CD45R-depleted

fraction as it drops from the flow restrictor (see Note 24).

11 If desired, the column can be washed and the retained CD45R+fraction can be

collected (see Note 25).

12 The purification process can be monitored by retaining an aliquot of the cellsprior to and after the separation These can then be labeled with fluorochrometagged antibodies and analyzed by flow cytometry Be aware that the CD45+cells are already labeled with FITC, so any labeling with CD11c will have toemploy an alternative fluorochrome

13 A repeat pass over a second column can again be used to increase purity

3.4 Isolation of Noncultured DC

In some circumstances culture of spleen cells overnight prior to separation

on metrizamide may be undesirable Here we present a method that employsthe immunomagnetic microbead technology discussed above to isolate spleen

DC without the need for culture

1 Prepare a single-cell suspension from spleen tissue using one of the methodsdescribed above

2 Resuspend spleen cells in miniMACS buffer at 400 µL of buffer per 108spleen cells

3 Add 100 µL of immunomagnetic beads coated with anti-CD11c (see Note 26)

4 Incubate for 15 min in the fridge

5 Wash in 5–10 mL cold miniMACS buffer by centrifugation at 200g for 10 min A

refridgerated centrifuge is preferable

6 Place the separation columns for separation: Use a MS+/RS+column washedthrough with 500 µL of buffer when working with less than 2 × 108total spleencells; use a LS+/VS+column washed through with 3 mL buffer for between 2× 108and 1×109spleen cells The smaller column can be used with either the miniMACS

or varioMACs system; the larger column will require the varioMACS system

7 Add the cell suspension to the top of the column(s) and allow the cells to enterthe column

8 Remove the unbound cells by washing the column with buffer: use 3×500 µL for

a MS+/RS+ column or 3 × 3 mL for a LS+/VS+ column

9 To remove bound CD11c+cells, remove the column well away from the magnet,add the appropriate volume to the top of the column (1 mL for MS+/RS+; 5 mLfor LS+/VS+), and flush out using the plunger provided with the column

10 Repeat the separation step using fresh columns (see Note 27).

4 Notes

1 2-Mercaptoethanol is an inhibitor of collagenase Therefore it should be omittedfrom medium used prior to the enzymatic digestion step if collagenase is used toproduce a spleen cell suspension

2 Collagenase D is recommended for maintenance of cell-surface protein integrity

3 There can be considerable batch-to-batch variation in collagenase Although aconcentration of 1 mg/mL usually gives satisfactory results, it may prove neces-sary to adjust this concentration

4 We use Falcon flasks Products from other manufacturers may also be suitable,but there could be variation in performance Small-tissue culture-grade Petridishes may also be used

5 Do not be tempted by the cheaper centrifugation grade metrizamide—it doesn’twork! We have no experience with metrizamide from other manufacturers

6 It is important to avoid air bubbles in the buffer, as these will affect the mance of the separation column Degassing of the buffer is recommended by thecolumns’ manufacturers At the very least the buffer should be prepared andhandled so as to mimimize frothing To this end we prepare our buffer well inadvance of use

perfor-7 If in doubt about the sterility of monoclonal antibodies or immunomagnetic beads,small volumes can be sterilized by centrifugation through 0.22 µm Spin-X cen-trifuge tube filters (Costar; cat no 8160) at full speed in a microfuge for 2 min.When using beads, be sure to resuspend the pellet that forms in the bottom of thetube All antibody concentrations stated are ones that we have found to work well

in general, but investigators may need to vary these for their own applications

8 “Fc-Block” is a mixture of unconjugated monoclonal antibodies to CD16 andCD32 (FcaIII/II), and, as its name suggests, it helps reduce nonspecific binding

of the labeling antibodies via Fc receptors

9 For best performance, precool the columns, magnets, and holders in a fridge orcold room We also use “cool packs” supplied in the packaging of many cooledproducts, to keep the apparatus cool during prolonged separations

10 If proceeding directly to preparation of cell suspensions from the spleen, it isbetter to keep the organ at room temperature than to expose it to the “shock” ofthe temperature changes involved in placing on ice and then warming up again insubsequent handling

11 The length of the incubation may need to be varied with different batches ofcollagenase

12 The performance of metrizamide gradients can be affected by small variations inosmolarity of the RPMI-1640 medium used to prepare them The osmolarity ofRPMI may vary slightly between manufacturers depending, for instance, onwhether the medium is intended for use primarily with human or mouse cells.The osmolarity suitable for mouse cells is required for successful enrichment of

DC If your gradients perform poorly, it may be worth switching suppliers ofRPMI

13 There is anecdotal evidence that metrizamide gradients perform better when theyhave been through one freeze-thaw cycle Therefore, we do not use freshly pre-pared metrizamide for separations

14 Do not be tempted to remove tissue fragments from the cell suspension beforeovernight culture These fragments are probably an important source of migrat-ing DC

15 Ideally use 10 mL of medium per spleen This amount will then be cultured intwo flasks and separated over two metrizamide columns If processing manyspleens, this can be reduced to 5 mL per spleen to reduce handling but do not betempted to reduce this volume further It is also inadvisable to “scale up” theseparation procedure by using bigger flasks and columns

16 The exact length of the “overnight” culture can influence the maturity and tion of the DC obtained Therefore, it is important to be consistent in the length ofthis incubation

func-17 Do not allow the metrizamide to become too warm This can be a problem innon-air-conditioned labs on hot summer days

18 The pellet can be used as a source of lymphocytes However, these cells need to

be handled gently to allow them to recover from exposure to the hypertonicmetrizamide

19 Sudden changes in DC yields can be a sensitive indicator of the presence ofinfection within an animal facility

20 The purity of DC separated on metrizamide varies with mouse strain Forinstance, we routinely obtain better purity of DC from the spleen of BALB/cmice than from C3H mice

21 To avoid frothing, resuspend gently using a pipet tip rather than by vortexing.

22 Incubation on ice is also possible but the time of incubation will need to beextended (20–30 min)

23 Wash thoroughly to ensure all EDTA is removed

24 Flow through the column is much slower when the flow restrictor is in place If

flow stops, restart by gently pushing the plunger supplied into the syringe barrel.

Push no more than is absolutely necessary to restart flow

25 With the flow restrictor in place, the purity of the retained cells may be reduced

To improve purity of the retained CD45R+population, a pass over a second umn with no restrictor in place may bring benefit

col-26 The volume of beads added can be reduced to 50 µL without appreciable loss onrecovery or purity of DC Further reduction is not recommended

27 When separating DC directly from whole spleen cells, this second pass over thecolumn is essential for good purity Expect up to 98% of the recovered cells to beMHC class II positive Of these cells, up to 95% express CD11c Expect torecover approx 2.5% of the starting spleen cell suspension.

References

1 Steinman, R M and Cohn, Z A (1973) Identification of a novel cell type inperipheral lymphoid organs of mice I Morphology, quantitation, tissue distribu-

tion J Exp Med 137, 1142–1162.

2 Steinman, R M and Cohn, Z A (1974) Identification of a novel cell type in

peripheral lymphoid organs of mice II Functional properties in vitro J Exp.

Med 139, 380–397.

3 Vremec, D and Shortman, K (1997) Dendritic cell subtypes in mouse lymphoidorgans Cross-correlation of surface markers, changes with incubation and dif-

ferences among thymus, spleen, and lymph nodes J Immunol 159, 565–573.

4 Crowley, M., Inaba, K., Witmer-Pack, M D., and Steinman, R M (1989) Thecell surface of mouse dendritic cells: FACS analyses of dendritic cells from dif-

ferent tissues including thymus Cell Immunol 118, 108–125.

5 De Smedt, T., Pajak B., Muraille, E., et al (1996) Regulation of dendritic cell

numbers and maturation by lipopolysaccharide in vivo J Exp Med 184,

1413–1424

6 Leenen, P J M., Radosevic, K., Voerman, J S A., et al (1998) Heterogeneity

of mouse spleen dendritic cells: in vivo phagocytic activity, expression of

mac-rophage markers, and subpopulation turnover J Immunol 160, 2166–2173.

7 Maldonado, L., De Smedt, T., and Michel, P., (1999) CD8alpha+ and CD8alpha–subclasses of dendritic cells direct the development of distinct T helper cells in

vivo J Exp Med 189, 587–592.

8 Suss, G and Shortman, K (1996) A subclass of dendritic cells kills CD4 T cells

via Fas/Fas-ligand- induced apoptosis J Exp Med 183, 1789–1796.

9 Vremec, D., Pooley, J., Hochrein, H., Wu, L., and Shortman, K (2000) CD4 and

CD8 expression by dendritic cell subtypes in mouse thymus and spleen J.

Immunol 164, 2978–2986.

From: Methods in Molecular Medicine, vol 64: Dendritic Cell Protocols

Edited by: S P Robinson and A J Stagg © 2001 Humana Press Inc., Totowa, NJ

Isolation of Mouse Thymic Dendritic Cells

Fabienne Anjuère and Carlos Ardavín

1 Introduction

The method described in this chapter for the isolation of mouse thymicdendritic cells (DC) is an optimization of our previously published methods

(1,2) and involves the following major steps:

1 Enzymatic digestion of thymic fragments with collagenase and DNase

2 Separation of a very-low-density cell fraction (VLDF)

3 Magnetic depletion of T-lineage cells, B cells, macrophages, and granulocytes

4 Positive selection of DCs by magnetic cell sorting (MACS)

This isolation method has been designed on the basis of the phenotype of

thymic DCs, which belong to the lymphoid DC lineage (3), and therefore are

positive for CD8 and CD11c, but negative for CD3, CD4, CD25, B220, Mac-1,

the macrophage antigen F4/80, and the granulocyte antigen Gr1 (4).

In contrast to previously described DC isolation methods and with theexception of the enzymatic digestion step, this isolation method is performedentirely at 4°C thereby avoiding prolonged incubation steps at 37°C, which

might alter the phenotypic and functional characteristics of DC (5) The method

involves the separation of a VLDF using an Optiprep (Nycomed Pharma AS)

centrifugation medium with a density of 1.055 g/mL This VLDF represents

0.2–0.4% of total thymocytes, i.e., 10 times less than the low-densitiy cell tions used in previous protocols The use of such VLDF makes the subsequentmagnetic bead depletion more efficient and less expensive

frac-Since DC represent around 0.1% of total thymocytes (4), a minimum

num-ber of thymuses are needed in order to obtain a reasonable numnum-ber of purified

3

DC The method described below is optimized for purifying DC starting from

20 4–6 wk-old mice, i.e., around 4.0× 109thymocytes The protocol for

scal-ing this method either up or down is explained in the Subheadscal-ing 3.2.

2 Materials

1 RPMI 1640 medium: RPMI 1640 medium supplemented with 1 mM sodium

pyru-vate and 100 U/mL penicillin–streptomycin Store at 4°C

2 Collagenase/DNase solution: Collagenase (0.5 mg/mL collagenase A,Boehringer-Mannheim) and DNase (40 mg/mL DNase I, grade II; Boehringer-Mannheim) in RPMI 1640 medium supplemented with 5% fetal calf serum (FCS).The collagenase/DNase solution has to be freshly made DNase can be stored at –

20°C at 1 mg/mL in 2:1 glycerol:H2O

3 PBS-EDTA-FCS: Phosphate-buffered saline solution (PBS) supplemented with

5 mM EDTA and 5% FCS Store at 4 °C (see Note 1).

4 Optiprep solution: The 1.055 Optiprep solution (density: 1.055 g/mL) is preparedfrom a ready made Optiprep solution (60% w/v Iodixanol in water; density 1.320g/ml; Nycomed Pharma AS) as follows: Mix V parts (chosen volume) of theOptiprep working solution with V1parts of the NaCl diluent following the for-mula: (V× D) + (V1× D1) = (V + V1) x 1.055 A V1 = V × 2.14 Where; D

= density of Optiprep working solution = 1.162 g/mL, D1 = density of NaCldiluent = 1.005 g/mL Example: add 21.4 mL (= 10 mL x 2.14) of NaCl diluent

to 10 mL of Optiprep working solution to obtain 31.4 mL of 1.055 Optiprepsolution

5 Optiprep working solution: Mix 1 part Optiprep with 1 part of NaCl diluent

6 NaCl diluent: NaCl 0.8% (w/v) in 10 mM Tricine buffer pH7.4 containing 5 mM

EDTA The 1.055 Optiprep solution can be stored at 4°C for up to 4 wk

7 mAb mixture: Prepare a monoclonal antibody (mAb) mixture containing CD3 (clone KT3-1.1), anti-CD4 (clone GK1.5), anti-IL-2R_ (clone PC61.5), anti-B220 (clone RA3-6B2), anti-macrophage antigen F4/80 (clone C1.A3-1), andanti-granulocyte antigen Gr1 (clone RB6-8C5) All the mAbs must be purified fromculture supernatants by affinity chromatography with Protein G Sepharose(Pharmacia Biotech) and used at 10 µg/mL in PBS-EDTA-FCS Make aliquots andstore at –20°C The aliquot in current use may be kept at 4°C for up to 3 wk.Prolonged refrigerator storage and repeated freeze–thaw cycles must be avoided

anti-3 Methods

1 Pool thymuses from 20 mice and cut into small fragments with blunt scissors inempty 50 mL polypropylene conical centrifuge tubes (Do not add any liquidmedium in this step)

2 Add 5 mL of collagenase/DNase solution and incubate for 10 min at 37°C withcontinuous agitation

3 Filter the digested fragments through a stainless-steel sieve with a 60-mesh screen(Sigma) and make a cell suspension in a 50-mL polypropylene conical centrifuge

tube by washing the digested tissue with 50 mL of RPMI 1640 medium ing 5 µg/mL DNase I (pre-warmed to 37°C) (see Note 2).

contain-4 Wash the cell suspension twice with 50mL cold PBS-EDTA-FCS containing 5mg/mL DNase I, and count For this and the following washing steps centrifuge

at 540g for 5 min at 4°C

5 Resuspend the cells in cold 1.055 Optiprep solution at 1.5× 108 cells/mL

6 Density gradient centrifugation is performed in 14 mL polypropylene bottom tubes (Falcon; cat no 2059) Using a Pasteur pipet, carefully layer 3 ml

round-of the cell suspension over 2 ml round-of 1.055 Optiprep solution and then layer 1 mL

of FCS above the cell suspension (see Fig 1) The 14 mL tubes must be

pre-cooled and kept at 4°C before layering the cell suspension (see Note 3); 8 to 10

tubes are generally needed when starting from 20 thymuses

7 Centrifuge at 1700g for 10 min at 4 °C and collect the VLDF (see Fig 1) with a

Pasteur pipet at the interface between the Optiprep and the FCS layers (select alow acceleration/deceleration rate centrifugation program)

8 Wash the VLDF three times with 15 mL PBS-EDTA-FCS in a 15 mL rene conical tube and then count the cells The VLDF constitutes a DC-enriched

polysty-fraction containing 15–20% DC (see Subheading 3.1 Fig 2).

9 Incubate the VLDF with the mAb mixture for 40 min at 4ºC Use 25µL of mAbmixture per 1× 106 VLDF cells

10 Wash the VLDF twice in 15 mL of PBS-EDTA-FCS

11 Resuspend the cells in 25µL of PBS-EDTA-FCS per 1 × 106VLDF cells, andkeep at 4°C while you wash the magnetic beads in order to remove the sodiumazide used as preservative

12 Wash the anti-rat Ig coated magnetic beads twice with cold PBS-EDTA (withoutFCS) in a 5 ml polystyrene tube (Falcon, cat no 2052) The beads are used at aratio of seven beads per cell Use a Magnetic Particle Concentrator (Dynal A.S.)

Fig 1 Preparation of density gradients for the generation of the DC enriched VLDF.

to retain the magnetic beads according to the manufacturer’s instructions.Remove the PBS-EDTA after the second wash.

13 Add the VLDF cell suspension to the 5 mL polystyrene tube containing theprewashed magnetic beads and mix gently

14 Transfer the VLDF/magnetic bead suspension to a 1 mL round-bottom pylene CryoTube vial (Nunc; cat no 375353) and incubate for 30 min at 4°Cwith gentle rotation (e.g., in a Dynal sampler mixer)

polypro-15 Add 1 mL of PBS-EDTA-FCS to the VLDF/magnetic bead suspension, mix gently

using a 1 mL micropipette, and transfer to a 5 mL polysterene tube (see Note 4).

16 Place the tube in a Magnetic Particle Concentrator and leave it for 4 min

17 Transfer the supernatant to a clean tube with a Pasteur pipet, taking care not todisturb the magnetic bead-rosetted cells collected at the tube wall, which must bediscarded The supernatant constitutes a highly DC-enriched fraction (HDCF)

containing 70–80% DC (see Subheading 3.1 Fig 2).

18 Incubate the HDCF with a biotin-conjugated anti-CD11c mAb (clone N418) at

10µg/mL for 20 min at 4°C Use 25 µL of mAb per 1 × 106 HDCF cells

19 Wash in PBS-EDTA-FCS

20 Incubate the HDCF with strepavidin-conjugated MACS microbeads (MiltenyiBiotec) diluted at 1/10 in PBS-EDTA-FCS for 10 min at 4°C, using 100 µL ofmicrobeads per 107 HDCF cells

21 Wash in PBS-EDTA-FCS and resuspend in 500 µL of PBS-EDTA-FCS

22 Purify the N418+DC from the HDCF by MACS, using MACS MS+or RS+tive selection columns (Miltenyi Biotec) in combination with a MACS separator(Miltenyi Biotec), following the manufacturer’s instructions After elution fromthe column the DC preparation has a purity > 97%

posi-Fig 2 Flow cytometric analyses of cell suspensions generated at different

stages of DC purification DC are identified as cells staining positively for bothCD11c and CD8 following labeling with monoclonal antibodies

3.1 Expected Results

Figure 2 shows the expected phenotypic profile (corresponding to a CD11c

vs CD8 immunofluorescent staining analyzed by flow cytometry) of the cellfractions obtained: after enzymatic digestion and centrifugation in Optiprep1.055 (VLDF); after depletion with magnetic beads (HDCF); after magneticcell sorting of N418+ cells (purified DCs)

The VLDF represents 0.2-0.4% of total thymocytes and contains around20% N418+cells Among N418+cells of the VLDF around 80% are DCs and20% are Mac-1+ F4/80+ thymic macrophages N418- cells in the VLDF corre-spond mainly to immature T-lineage cells and thymic B cells

The HDCF represents 15–20% of the VLDF and contains 70–80% of N418+DCs, the remaining N418- cells being essentially immature T-lineage cells.Excluding DCs, this cell fraction is therefore devoid of other antigen present-ing cells, such as macrophages or B cells and could be used as an enriched-thymic DC fraction for certain purposes Finally, N418+ cells represent >97%

of MACS-purified DCs

The yield of the isolation method reported here is 40–80× 103purified DCsper thymus of a 4-6 wk-old mouse

3.2 Scaling the Isolation Method

Increase or decrease proportionately to the starting number of thymuses/thymocytes the quantity of the reagents and the number of tubes required, tak-ing into consideration the following points: It is not recommended to start theisolation of thymic DCs with less than 2 x 109 thymocytes, i.e., around 10thymuses of 4–6 wk-old mice It is highly recommended that the 14 mLpolypropylene tubes, the 5 mL polystyrene tubes, and the 1 mL Cryotubes

specified in the Subheading 3 should be used, although other high-quality

tubes with similar characteristics should also be adequate Do not modify theconditions specified for obtaining the VLDF in terms of both the cell concen-

tration and the volume corresponding to the different layers In Subheading

3., step 14 do not use more than 15× 106VLDF cells and the corresponding

magnetic beads per 1 mL Cryotube Finally in Subheading 3., step 15 use one

5 mL polystyrene tube per each 1ml Cryotube required

prewarmed in order to avoid the formation of cell aggregates, which could greatlyreduce the yield of the method.

3 The 14 mL polypropylene tubes used for obtaining the VLDF must be precooled

in order to facilitate the setting of the discontinuous Optiprep gradient

4 Mixing the PBS-EDTA-FCS with the VLDF magnetic bead suspension is a cal step in this isolation method It must be done gently but efficiently in order toavoid the detachment of some contaminating cells bound loosely although spe-cifically to the beads and on the other hand to disrupt the aggregates which areusually formed during the previous incubation

criti-References

1 Vremec,D., Zorbas, M., Scollay, R Saunders, D J., Ardavin, C F., andShortman, K (1992) The surface phenotype of dendritic cells purified frommouse thymus and spleen: Investigation of the CD8 expression by a subpopula-

tion of dendritic cells J Exp.Med 176, 47–58.

2 Ardavin, C., Waanders, G., Ferrero, I., Anjuere, F., Acha-Orbea, H., andMacDonald, H R (1996) Expression and presentation of endogenous mousemammary tumor virus superantigens by thymic and splenic dendritic cells and B

cells J Immunol 157, 2789–2794.

3 Ardavin,C., Wu, L., Li, C.-L., and Shortman, K (1993) Thymic dendritic cellsand T cells develop simultaneously in the thymus from a common precursor popu-

lation Nature 362, 761–763.

4 Ardavin, C (1997) Thymic dendritic cells Immunol Today 18, 350-361.

5 Wu, L., Vremec, D., Ardavin, C., Winkel, K., Suss, G., Georgiou, H.,Maraskovsky, E., Cook, W., and Shortman, K (1995) Mouse thymus dendriticcells: kinetics of development and changes in surface markers during maturation

Eur J Immunol 25, 418–425.

29

From: Methods in Molecular Medicine, vol 64: Dendritic Cell Protocols

Edited by: S P Robinson and A J Stagg © 2001 Humana Press Inc., Totowa, NJ

Isolation of Dendritic Cells from Rat Intestinal

Lymph and Spleen

Gordon MacPherson, Michelle Wykes, Fang-Ping Huang,

and Chris Jenkins

1 Introduction

1.1 Lymph Dendritic Cells

Dendritic cells (DC) are rare cells in peripheral tissues, and their isolationfrom tissues is fraught with problems Thus, the proportion of DC within atissue that is extracted is unknown, isolation procedures may select for sub-populations, and the isolation procedure itself may affect their properties Aspart of their life history, DC migrate from peripheral tissues, via peripheral,afferent lymph to lymph nodes, even in the absence of exogenous antigenicstimulation They are extracted within the node and very few, if any, appear inefferent lymph These lymph DC (L-DC) represent a population that hasmatured in the periphery, that may have acquired antigen (Ag), and that may

be engaged in active Ag transport to lymph nodes As such they are a ologically relevant DC population In large animals such as sheep and cattle,L-DC can be isolated by direct cannulation of peripheral lymphatics, but yieldsare relatively low In rodents, direct cannulation of some peripheral lymphatics

physi-is possible (1), but yields of cells are minuscule To get around thphysi-is problem we and others (1–7), have utilized lymphadenectomy as a means of collecting

pseudo-afferent lymph When lymph nodes are removed, over a period ofweeks, the afferent and efferent lymphatics join as part of the healing process,leaving cells in peripheral lymph free to enter central lymph Central lymphat-

ics are relatively easy to cannulate, and cannulation can be maintained for

con-siderable periods of time (see Note 1).

This approach to L-DC isolation has the major advantages that DC can becollected under near-physiological conditions, that they can be collected freshwithout any complicated manipulations, and that they can be metabolically

“frozen” by collecting them into cooled vessels It has the disadvantages thatonly DC at a particular stage of their life history can be collected, and that in

the rat at least, only DC from the small intestine or liver (8,9) can be collected.

The isolation of DC by these techniques involves two stages, in the first, thelymph nodes draining the relevant tissue bed are removed The animal is thenleft for a period of weeks to permit healing of afferent and efferent lymphatics,

and then the thoracic duct is cannulated using Gowans’ modification (10) of the original Bollman et al method (11).

1.2 Spleen Dendritic Cells

The first DC to be isolated were derived from murine spleen (12) Isolation

involved enzymatic digestion of the spleen, buoyant density separation of lightcells, short-term adherence, overnight culture, and removal of FcR+cells Thisgave a population of DC that has been used as the standard for many years

Similar methods were used to isolate DC from rat spleen (13) It has become

clear, however, that there is more than one population of DC in murine spleen

(14), that the original isolation procedure was selective for 33D1+DC foundmainly in the marginal zone and possibly red pulp, and that the classical inter-

digitating cells (IDC) were not being extracted (15) A modification of the

original technique involving the use of EDTA was needed to extract IDC ciently Another problem that needs to be considered is the effect of the isola-tion procedure itself on the phenotype and function of the isolated DC Thus it

effi-is clear that the properties of murine Langerhans cells (16), murine heart and kidney DC (17), and rat intestinal lymph DC (18) are markedly changed by

short term in vitro culture Recently, it has been shown that splenic DC isolated

without overnight culture display characteristics typical of immature DC that

are not found in DC isolated by standard methods (19) Thus, when isolating

DC from any tissue, including the spleen, careful consideration must be given

to the possible selectivity of the isolation procedure and its effects on the

resulting DC (see Note 2).

In this chapter we describe the procedure that we use to isolate fresh DCwithout overnight culture This has become our standard method as we con-sider that it provides DC that are closer to in vivo DC Rat DC can also be

prepared using the original method described for murine splenic DC (12).

2 Materials

2.1 Lymph Dendritic Cells

1 Specific pathogen-free rats around 6 wk of age are used for surgical

lymphadenec-tomy (see Note 3).

2 Anesthetic apparatus: Any small animal apparatus suffices, but we use a othane anaesthetic system (International Market Supply, Congleton, UK) No pre-operative treatment is required

hal-3 Hair clippers (International Market Supply)

4 Surgical instruments including scalpel, scissors and dagger forceps (Holborn 4.Surgical Instrument Co., Margate, UK)

5 Sutures including absorbable 4/0 sutures (Ethicon) and needles (John Weiss,Milton Keynes, UK)

6 Operating light (Cold Source Fibre Optic Lamp, Schott Scientific LaboratorySupplies, Nottingham UK)

7 Operating board (see Note 4).

8 Heparin sodium (CP Pharmaceuticals Ltd, Wrexham UK)

9 70 µm cell strainers (Falcon 2350, Becton Dickinson)

10 Washing buffer: 2–5% fetal calf serum (FCS) (Gibco-BRL, Paisley, Scotland)

Mix: 20 parts stock A, 5 parts stock B, 5 parts stock C, and 70 mL distilled water

12 Counting chambers: Fast Read 10 chambers (Immune Systems, Paington, UK)

13 NycoPrepTM 1.068 density medium (NYCOMED, Oslo, Norway)

14 Monoclonal antibodies: OX52 (Pan T cell), OX19 (CD5), OX8 (CD8), OX12 (Igkappa-chain), OX33 (B cell CD45), OX62 (rat DC) Commercial suppliersinclude Serotec, Kidlington, UK and The Binding Site, Birmingham, UK.

15 Goat anti-mouse IgG labelled with immunomagnetic microbeads (MiltenyiBiotec, Order No 484-02, Bergisch Gladbach, Germany)

16 MACS buffer: 1% Bovine Serum Albumin (BSA) (Sigma), 2mM EDTA, and0.01% sodium azide in PBS

17 Normal rat serum (heat-inactivated, 56°C, 30 min)

2.2 Spleen Dendritic Cells

1 Specific pathogen-free rats of any strain, at 10-12 weeks of age

2 Sterile Petri dishes

3 Curved forceps and straight forceps with serrated ends

4 Hanks balanced salt solution (HBSS) with Ca++, Mg++

5 HBSS without Ca++, Mg++

6 Enzyme cocktail: 2mg/ml Collagenase D (Boehringer Mannheim) and 0.5mg/mlDNAase (Boehringer Mannheim) in HBSS (with Ca++, Mg++) Keep on ice andfilter sterilise just before use Stock solutions of collagenase D (8mg/ml in HBSSwith Ca++, Mg++) and DNAase (0.5mg/ml) can be stored frozen until required

(see Note 5).

7 Collection buffer: 20mM EDTA in HBSS (without Ca++, Mg++) The EDTAinhibits the enzyme activity by chelation of Ca++ Keep this buffer on ice atall times

8 Gey’s Solution for RBC lysis See Subheading 2.1.

9 I-5 medium: Iscove’s modified Dulbecco’s medium supplemented with 50mg/mlpenicillin, 50mg/ml streptomycin, 2 mM glutamine, 5% foetal bovine serum(FCS) and 5x10-5M 2-`-mercaptoethanol Medium and supplements from GibcoBRL, supplied by Life Technologies Ltd, Paisley, UK

10 Primary and secondary antibodies: OX52 (pan T cell), OX8 (CD8, T cells and

NK cells), OX 12 (anti-Ig light chain ), OX33 (pan-B cells) (Serotec).Biotinylated anti-Ig µ chain and anti-Ig a chain antibodies (The Binding Site).Rabbit anti-rat IgG, IgM and IgA (absorbed for mouse Ig and FCS, Dako Z0494)and rabbit anti-mouse Ig (Dako Z0456)

11 Sheep red blood cells (SRBC) (TCS Microbiology, Botolph Claydon,UK)

12 Histopaque, density = 1.083 (Sigma)

13 Dynal beads coated with anti-mouse Ig and streptavidin

14 Sterile, short cut Pasteur pipet

3 Methods

3.1 Lymph Dendritic Cells

3.1.1 Mesenteric Lymphadenectomy

We routinely remove mesenteric nodes surgically, but it also has been shown

that freezing the nodes leads to immediate loss of their ability to retain DC (20).

1 Anaesthetize the rat and clip the hair from the abdomen Tape the feet to theoperating board and swab the abdomen with 70% ethanol.

2 Make a mid-line incision with a scalpel from the xiphisternum for about quarters of the length of the abdomen Incise the muscle layers along the mid-linewith scissors

three-3 Gently reflect the intestine to the right (rat’s left side) and support it on gauzesoaked in saline The chain of mesenteric nodes is clearly visible at the root of themesentery, close to the inferior vena cava

4 Starting at the anterior end of the chain, pick up a node with forceps Use anotherpair of forceps to tear away the surrounding connective tissue and remove thenode Press with a cotton bud to stop any minor bleeding that occurs Repeat untilthe whole chain has been removed

5 Check hemostasis and replace the intestines

6 Suture the abdominal wall in layers with interrupted sutures Suture the musclelayers with an absorbable suture and the skin with silk (clips can be used for theskin incision)

7 Postoperative recovery is usually quick and uneventful Infection is very rare,and the only other complication of which we are aware is postoperative ileus,again very rare

8 Rest the rats for at least 6 wk before thoracic duct cannulation to permit healing

of the lymphatics

3.1.2 Thoracic Duct Cannulation

The technique of thoracic duct cannulation is described in detail in the

fol-lowing and will not be repeated here (see Note 1):

1 Ford W.L (1978) “The preparation and labelling of lymphocytes,” in Handbook

of Experimental Immunology, Ed Weir, Blackwell, Oxford, Chapter 23

2 Waynforth, H B and Flecknell, P A (1992) Experimental and Surgical

Tech-nique in the Rat, Academic Press, London, pp 264–268.

3.1.3 Lymph Collection and Cell Preparation

1 Collect lymph for suitable periods into flasks containing heparinized (20 U/mL)

PBS cooled on ice We do not normally collect for more than 20 h at a time (see

4 Centrifuge at 400g for 8 min, resuspend the cells in 1 mL medium, and removeany contaminating RBC by lysis in Gey’s solution Add 4 mL Gey’s solution andincubate for 2 min on ice

5 Wash the cells twice with cold washing buffer, count in a hemocytometer, and

finally resuspend in the same buffer at about 2 × 107cells per mL Typical DCcan be recognized in the counting chamber by their size and irregular outline.Usually, about 3–5 × 108total cells per rat are obtained from a 20 h lymph collec-tion, of which DC form about 0.3–0.5% of the total cells The dominating celltypes are T and B lymphocytes and, in some cases, contaminating RBC There-fore, procedures that enable enrichment of DC are necessary for most studies.Depending on the purpose of the experiment and the purity required, DC can beenriched and, when necessary, further purified by using combinations of themethods described below.

3.1.4 Enrichment of L-DC by Density Centrifugation

DC have a lower average density than lymphocytes However, the densities

of the two cell types overlap, especially DC with large lymphocytes (blasts) It

is difficult to separate them effectively based on density differences alone Weroutinely use NycoPrepTM 1.068 (see Note 7).

1 Layer 4 mL of the DC suspension prepared above over 3 mL of the NycoPrepTM

1.068 solution in a 12-mL clear centrifuge tube (see Note 8).

2 Centrifuge the cells at 600g for 15–20 min at room temperature An interface

that contains DC should be visible and at the bottom of the tube is a cell pellet

that consists mainly of lymphocytes (see Note 9).

3 Pour carefully and combine all cells in the suspension from three tubes into one50-mL centrifuge tube and top up with washing buffer (at least double the origi-nal volume) Discard the cell pellets

4 Centrifuge the cells at 600g for 8 min at 4oC to remove the density medium

Wash the cells twice more with washing buffer, centrifuging at 400g for 5 mins,

and count At this stage, DC normally represent 20–40% of the total recovered

cells (see Note 10) The contaminating cells are small and in particular large lymphocytes (see Note 1).

DC can be further enriched by repeating the above steps, which normallydoubles the DC purity (up to 60% can be achieved) Alternatively, DC in thepartially enriched preparation may be further purified by using one of the fol-lowing methods to positively select DC or by negatively selecting the remain-ing lymphocytes

3.1.5 Purification of L-DC by Immunomagnetic Separation

In our lab, we routinely use positive or negative separation using the MACS

system as a second step for purifying L-DC after NycoPrep enrichment (see

Notes 11 and 12).

3.1.5.1 NEGATIVE SELECTION

DC are enriched by negative selection of T and B cells We use a tion of monoclonal antibodies OX52 (pan T), OX19 (CD5), and OX8 (CD8) to

combina-deplete T cells; and a combination of OX12 (kappa-chain) and OX33 (B cellCD45) to deplete B cells.

1 Incubate NycoPrep-enriched cells (Subheading 3.1.4) at 4°C for 20 min with amixture of equal volumes of the above antibodies as tissue culture supernatants(TCS), supplemented with (optional) additional purified OX52 and OX12 anti-bodies at 10 µg/mL Up to 107total cells per ml of the antibody mixture can beefficiently labeled in this way for separation

2 Wash the cells three times with cold washing buffer

3 Incubate the cells with the MACS beads labeled with antibody, 1:5 diluted in theMACS buffer and used at 0.1 mL per 107cells, for 15 min at 4°C 2–5% of heat-inactivated normal rat serum can be added in the antibody diluent to reduce non-specific binding or crossreactivities

4 Wash the cells twice with the washing buffer and then once in the MACS buffer

5 Carefully resuspend the cell pellets in a small volume of degassed-MACS buffer,0.5 mL per (or up to) 108total cells They are now ready for cell separation on the

MACS columns (see Note 12 and follow the manufacturer’s instructions).

6 Details of yield and recoveries are given in Note 13.

3.1.5.2 POSITIVE SELECTION

To positively select L-DC, we use a mouse rat DC monoclonal body, OX62 (IgG1) (See Note 11), which is used at 10 µg/mL or as neat TCS.The staining procedure is the same as that described for the negative selection

anti-(Subheading 3.1.5.1) Other methods of purifying L-DC are described in Note 14.

3.1.6 Conclusions

Lymph DC, freshly collected, represent cells that are as close to in vivo DC

as is at present possible to obtain Their isolation is not, however, technicallystraightforward, requiring specialized surgical skills and equipment Theyare difficult to obtain in large numbers and represent only one stage in the lifehistory of DC from one tissue However, by carefully following the procedure

as described above, DC that are “near-physiological” can be obtained with goodyields and high purity

3.2 Spleen Dendritic Cells

3.2.1 Digestion of Spleens

1 Excise spleens from 10–12 wk-old rats with sterile instruments and place in a 3.5

cm sterile Petri dish with 3 mL of enzyme cocktail

2 Fill a sterile 5 mL syringe with 2–3 mL enzyme, and using a 23G needle injectthe enzyme into several sites in the spleen When inserting the needle into thespleen, use sterile forceps to hold the tissue in place and pierce the tissue withcaution so as not to exit the other side Areas surrounding the injection site appear

as lighter colored patches Repeat several times until a large portion of the spleen

has been flushed of loose cells (see Note 15).

3 Tear the spleen into small pieces using two sterile forceps, and incubate at 37°Cfor 15 mins After this, carefully remove the free cell suspension using a sterileshort Pasteur pipet, put into collection buffer, and keep on ice Add more enzyme

to the remaining tissue pieces and “tease” the tissue This involves holdingthe tissue with the straight serrated forceps and scraping the sides of the curvedforceps against the tissue Incubate for 15 min at 37°C

4 Using a short pasture pipette, take up and release the tissue/cells mixture severaltimes This process will release cells from remaining tissue pieces Transfer thereleased cells to the collection buffer on ice If required, add remaining enzymeand repeat the incubation/ release process until no tissue pieces are visible

5 When the digestion is complete, pellet the cells at 400g for 10 min, and lyse red

blood cells using Gey’s (subheading 3.1.2., item 4) or a similar solution Wash

cells in I-5, resuspend in this medium, and incubate for 2–5 min on ice Debrisshould settle during this time Transfer the cell suspension to a fresh tube, leav-ing behind the debris

6 Pellet the cells at 400g for 5 min and incubate with the antibody cocktail (OX52,

OX8, OX12, OX33) for 30–60 mins at 4°C

7 During this incubation prepare 2 separate batches of SRBC, one coated with bit anti-rat IgG, IgM, and IgA (to deplete B cells) and the other with rabbit anti-mouse Ig (to deplete all antibody-coated cells) as described in Chapter 18.3.2.2 Removal of Non-dendritic Cells Using Rosetting and Dynal BeadsRosetting has several advantages over other methods such as the use ofDynal beads alone The method is relatively inexpensive, it removes macroph-ages via FcR binding, and the gradient step removes dead cells

rab-1 Wash antibody-coated spleen cells twice in PBS or I-5 and resuspend with theanti-rat Ig-coated SRBC After 2–3 min at room temperature, add the other batch

of SRBC (rabbit anti-mouse Ig-coated), mix, aliquot into small sterile vials androtate for 30 min at 4°C (see Note 16).

2 Layer onto the Histopaque (Sigma) density gradient medium and centrifuge for

20 min at 400g Collect the cells at the interface and wash in I-5.

3 Resuspend the cells in 5 mL I-5 and count Add four anti-mouse Ig andstreptavidin-coated Dynal beads/cell (stock concentration is 4× 108beads/mL)and incubate for 30 min at 4°C, mixing occasionally Place the tube on a Dynalmagnet for 3 min, remove unbound cells to a fresh tube, and place on the magnetagain Repeat three or four times until all the magnetic beads have been removed

4 Pellet the remaining cells and resuspend in I-5

3.2.3 Expected results

This method consistently yields from 7× 106to 107DC per rat spleen (see

Note 17) The cells are routinely characterized by flow cytometry and

immu-nocytochemistry, and contain at least 85% MHC class II+cells with less than

1% contaminating T or B cells The level of surface MHC class II expression isvery low on most cells and immunocytochemical labeling of cytospins showsthat a large portion of the MHC class II is intracellular Cytospins stained forimmunoglobulin show the absence of plasma cells The cells do not phagocy-tose opsonized sheep red blood cells indicating that they are not macrophages.

It should also be noted that only a small proportion of the cells shows the sic dendritic morphology immediately after isolation However, after overnightculture of these cells, 95–99% of the cells will express high levels of surface

clas-MHC class II, and the dendritic processes are more conspicuous (see Note 18).

4 Notes

1 It should be pointed out that thoracic duct cannulation requires considerable gical skill and specialized equipment for anesthesia and restraint of the cannu-lated animal The techniques involved are best learned in a laboratory where theyare already in use Lymph can be collected for periods of days; we routinelycollect for up to 2 days We have found that if lymph is collected for more than 2

sur-d, the proportion of B blasts increases, and they tend to co-purify with the DC

2 It is becoming apparent that in vitro manipulation of DC, even for short periods,can alter their phenotype and function This needs to be taken into account whentrying to relate in vitro and in vivo DC properties

3 Young rats are used because the nodes are relatively free of surrounding fat at thisstage and are easily visible We use males where possible, because at the time ofcannulation relatively little fat surrounds the thoracic duct The optimal time forcannulation is when the rats are about 12-wk-old Cannulation is possible at laterstages, but the increased amounts of fat around the duct increase the technicaldifficulty of the procedure

4 It may be advantageous to use a heated operating table, but this is not essential

It is essential that animals are kept warm after the operation until they are fullyrecovered

5 Collagenase D is used in preference to collagenase A We find that collagenase Dgives better viability without a large decrease in yields Batches of collagenase Avary in terms of both the viability and numbers of DC that are recovered

6 Twenty hours represent an overnight collection, allowing a whole day for cellmanipulations

7 NycoPrepTM 1.068 is a density medium with an adjusted osmolality and isslightly hypertonic Because lymphocytes are more sensitive than DC to theincreased osmolality, they dehydrate and become even denser resulting in muchimproved separation upon centrifugation We find that cell yields and viabilityare better with NycoPrep than with Metrizamide It is important not to overloadthe tube with cells; if large numbers are used, “streaming” will occur and DC will

be dragged into the density medium One of the major problems with DC tion is that DC form clusters with both T and B cells The use of EDTA helps tobreak up clusters and keeping the cells cold at all times is essential

separa-8 Clear centrifuge tubes are essential to allow visualization of the interface.

9 It is possible that some DC will end up in the pellet but there are probably veryfew; when we use pelleted cells to stimulate an allogeneic MLR, we get little or

no response

10 DC are counted in a hemocytometer chamber We visualize cells using Nomarskiinterference optics DC are identified by their size and irregular outline Countsdone this way give similar results to those done on cytospins At the end of longseparation procedures, particularly if they are carried out in the cold, DC may nothave the typical irregular outline and are thus difficult to identify If they are left

at room temperature in the hemocytometer for 10–15 min, they will regain thedendritic appearance

11 The choice of negative or positive selection depends on the intended application.One of the advantages of using negative selection is that DC negatively selectedcan easily be phenotyped using cell surface markers without interference fromantibodies used for the cell separation which remain bound to DC This is also areason for purifying DC that are to be used for functional assays when antibodiesused are likely to affect DC functions In addition, the negatively selected DCscan be subjected directly to further separation of DC into sub-populations bysubsequent positive selection

12 This note deals with the MACS separation procedure The major problems thatmight be encountered are :

Cell loss: Since the frequency of L-DC is very low and the cell separation

proce-dure is rather complex, poor cell yields potentially are a major problem Duringevery step of the cell separation procedures, significant cell loss is likely to occur.This can be caused by multiple factors including incorrect cell loading and wash-ing, cell adhesion or aggregation, cell trapping in the MACS columns, and so on

In our experience, it is important that: (1) all buffers used should be filteredand, for the MACS buffer, degassed; (2) cells should be kept cold throughoutand, if possible, the MACS columns should be precooled and the separation pro-cedures performed in a cold room; (3) during the NycoPrep procedure, do notover load cells; (4) during cell washing, as DC are generally less dense, sufficient

g force should be used to ensure that cells are adequately pelleted and then

resus-pended without delay (do not leave DC in the cell pellet longer than necessary).When washing cells after the density separation step, add a sufficient volume for

dilution of the samples which are in density medium, and use extra g force to

bring down cells during centrifugation; (5) resuspend cells thoroughly beforeloading onto the MACS column but try to avoid forming bubbles

For general cell separation purposes, there are two types of tion Columns (AS, BS, CS, and DS, for negative selection) and Positive Selec-tion Columns (XS+, VS+ and RS+) For isolation of DC, however, we found that

columns—DC with their long processes are likely to be trapped nonspecifically in the tion Columns, probably because this type of column has a matrix made of a steelfiber material In our experience, using the Positive Selection Columns (whichcontain spherical metal beads) for negative selection results in less DC trapping

Deple-despite this type of column having a smaller pore size (30 µm) than the Depletioncolumns (100 µm) However, the DC purity obtained this way may be slightlylower, possibly because this type of column has a lower magnetic strength,allowing weakly labeled cells to escape.

Cell impurity: Since DC are more likely to form aggregates and to be lost

during washing when an insufficient g force is used, low DC purity is alwaysassociated with significant DC loss In addition, owing to the long proceduresinvolved in DC separation in the cold (i.e., on ice), identifying DC morphologi-cally can be more difficult, especially for small DC Leaving the cells in thecounting chamber or slides at room temperature for 15 min before counting mayhelp Alternatively, immunostaining or FACS analysis of the cells using anti-body cell markers gives a better idea of cell purity

13 Usually, from one lymphadenectomized, cannulated rat, between 0.5 and 2× 106

DC can be obtained in a 20 h lymph collection A high purity of DC (> 90% byFACS staining) can be achieved by combining the NycoPrep and MACS (nega-tive selection) methods The purities and yields of the L-DC obtained very muchdepend on the methods used and the handling of cells Cell loss may be signifi-cant, especially when using the MACS separation procedure DC purity can bechecked by FACS using anti-DC markers, such as OX62 and OX6 (Class IIhi), or

by their morphological characteristics on slides or cytospin slides

14 As alternatives to immunomagnetic separation DC can be further enriched byrosetting or the use of Dynal beads These methods have been used for separation

of DC (21), and although we now routinely use MACS, rosetting may give higher

recoveries Positive selection by rosetting or Dynal beads has the disadvantagethat the SRBC or beads remain attached to the DC In the case of rosetting, DCcan be recovered by lysis of the SRBC, but we are not aware of a method ofremoving beads (“Detachabead” is not applicable.)

15 We find that direct injection of the spleen prior to mincing it gives higheryields of DC

16 We use a wheel that rotates at 20–30 rpm

17 The proportion of DC that can be isolated from any organ or tissues is uncertain.Our experience with spleen, lymph node, and small intestine has shown that arelatively small proportion is isolated but we have no quantitative data We havecompared collagenase digestion with mechanical isolation procedures and findthat the yield of DC is markedly (two or three times) greater with collagenase.The method we describe is slightly more laborious than the method involvingovernight adhesion, but the yields are higher and the DC are not “matured” byovernight culture, and thus are closer to in vivo DC

18 The method we have described mainly yields immature DC The majority ofMHC class II molecules is intracellular, but they will move to the surface if givenmaturation signals, including overnight culture The endocytic capacity of thecells is maximal during the first few hours after isolation but decreases rapidlywith culture If the cells are to be pulsed with antigen, this must be done soonafter isolation

1 Pugh, C W., MacPherson, G G., and Steer, H W (1983) Characterization of

nonlymphoid cells derived from rat peripheral lymph J Exp Med 157, 1758–79.

2 Mayrhofer, G., Holt, P G., and Papdimitriou, J M (1986) Functional

character-istics of the veiled cells in afferent lymph from the rat intestine Immunology 58,

379–387

3 Harkiss, G., Hopkins, J., and McConnell, I (1990) Uptake of antigen by afferent

lymph dendritic cells mediated by antibody Eur J Immunol 20, 2367–2373.

4 Bujdoso, R., Hopkins, J., Dutia, B M., Young, P., McConnell, I (1989)Characterisation of sheep afferent lymph dendritic cells and their role in antigen

carriage J Exp Med 170, 1285–1302.

5 Bujdoso, R., Harkiss, G., Hopkins, J., and McConnell, I (1990) Afferent lymph

dendritic cells: a model for antigen capture and presentation in vivo Int Rev.

Immunol 6, 177–86.

6 Howard, C J., Sopp, P., Brownlie, J., Parsons, K R., Kwong, L S., and Collins, R A.(1996) Afferent lymph veiled cells stimulate proliferative responses in allogeneic

CD4+ and CD8+ T cells but not gamma delta TCR+ T cells Immunology 88, 558–64.

7 Howard, C J., Sopp, P., Brownlie, J., Kwong, L S., Parsons, K R., and Taylor,

G (1997) Identification of two distinct populations of dendritic cells in afferent

lymph that vary in their ability to stimulate T cells J Immunol 159, 5372–5382.

8 Matsuno, K., Ezaki, T., Kudo, S., and Uehara, Y (1996) A life stage of laden rat dendritic cells in vivo: their terminal division, active phagocytosis, and

particle-translocation from the liver to the draining lymph J Exp Med 183, 1865-1878.

9 Kudo, S., Matsuno, K., Ezaki, T., and Ogawa, M (1997) A novel migration way for rat dendritic cells from the blood: hepatic sinusoids-lymph translocation

path-J Exp Med 185, 777–784.

10 Gowans, J L (1959) The recirculation of lymphocytes from blood to lymph in

the rat J Physiol 146, 54.

11 Bollman, J L., Cain, J C., and Grindley, J H (1948) Techniques for the

collec-tion of lymph from the liver, small intestine or thoracic duct of the rat J Lab.

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12 Steinman, R M., Cohn, Z A (1973) Identification of a novel cell type in

periph-eral lymphoid organs of mice I Morphology, quantitation, tissue distribution J.

Exp Med 137, 1142–1162.

13 Klinkert, W E F., LaBadie, J H., and Bowers, W E (1982) Accessory andstimulating properites of dendritic cells and macrophages isolated from various

rat tissues J Exp Med 156, 1–19.

14 Metlay, J P., Witmer-Pack, M D., Agger, R., Crowley, M T., Lawless, D., andSteinman, R M (1990) The distinct leukocyte integrins of mouse spleen den-

dritic cells as identified with new hamster monoclonal antibodies J Exp Med.

16 Schuler, G., and Steinman, R M (1985) Murine epidermal Langerhans cells mature

into potent immunostimulatory dendritic cells in vitro J Exp Med 161, 526–546.

17 Austyn, J M., Hankins, D F., Larsen, C P., Morris, P J., Rao, A S., Roake, J A.(1994) Isolation and characterization of dendritic cells from mouse heart and kid-

markers, and subpopulation turnover J Immunol 160, 2166–2173.

20 Gyure, L and Hall, J (1990) A quick method for obtaining lymph borne dendritic

macrophages from rats J Immunol Methods 131, 49–53.

21 Liu, L M., Zhang, M., Jenkins, C., and MacPherson, G G (1998) Dendriticcell heterogeneity in vivo: Two functionally different dendritic cell populations

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43

From: Methods in Molecular Medicine, vol 64: Dendritic Cell Protocols

Edited by: S P Robinson and A J Stagg © 2001 Humana Press Inc., Totowa, NJ

Isolation, Enrichment, and Culture of Murine

Epidermal Langerhans Cells

Franz Koch, Eckhart Kämpgen, Gerold Schuler,

and Nikolaus Romani

1 Introduction

Langerhans cells are the epidermal variant of the dendritic cell system

(1–5) They were—unknowingly, though—the first dendritic cell to be described:

In 1868 Paul Langerhans published his observations of a dendritically shaped

cell in the human epidermis (6) Until the early 1990s (i.e., the advent of

meth-ods for mass production of dendritic cells from blood or bone marrow) hans cells served as a prototype dendritic cell The basis of our currentknowledge on the maturation of dendritic cells stems from experiments with

Langer-epidermal Langerhans cells (7–9).

Still, Langerhans cells remain interesting to the researcher First, hans cells are of importance in many dermatologic diseases and their role inthese processes needs to be elucidated A typical example is contact hypersen-

Langer-sitivity (10,11) Second, Langerhans cells may serve as a model to learn about

the heterogeneity of dendritic cell populations A recent example is the finding

of Caux’s group that “non-Langerhans dendritic cells” derived from CD14+/CD1a– precursors in cord blood have different functions as compared to

“Langerhans type dendritic cells” derived from CD14-/CD1a+ precursors in

cord blood (12) Third, isolated Langerhans cells represent probably the

“cleanest” experimental system to study features of immature vs mature dritic cells In other words, in populations of freshly isolated Langerhans cells,the great majority of Langerhans cells are immature (> 95%); after short-term