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DC from Mouse Lymph Nodes 3
1
Isolation of Dendritic Cells from Mouse Lymph Nodes
Dmitry 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 these
cells in normal and pathological conditions. Therefore, lymph nodes are popu-
lar sites for the isolation of dendritic cells. Dendritic cells isolated from lymph
nodes represent “interdigitating” DCs that are localized in T-dependent regions
of lymph nodes. DCs represent about 1% of the total population of lymph node
cells. Therefore, in order to perform almost any functionaltests, the DC fraction
should 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 sepa-
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%) of
macrophages. The choice of lymph nodes is dependent on the purpose of the
experiment. The most commonly used lymph nodes are axillary, inguinal, and
popliteal. DCs can be further enriched using monoclonal antibodies and flow
cytometric cell sorting, magnetic beads separation, panning, or cytotoxic elimi-
nation with complement. All these methods are based on the negative selection
of DCs using anti-T, anti-B, and anti-macrophage antibodies. Since the first
step of isolation involves gradient centrifugation, granulocyte contamination
is negligible and further purification steps do not require use of anti-granulo-
cyte antibodies.
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
3
4 Gabrilovich
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 mL
syringes, and 5mL 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 com-
monly use a combination of penicillin, streptomycin, and Fungizone (antibiotic–
antimycotic, Gibco-BRL). DMEM can be used without serum. RPMI 1640 should
be supplemented with 10% FCS (RPMI-FCS).
4. Metrizamide gradient. Dissolve 7.25g metrizamide with 45mL RPMI in a 50-mL
tube. It usually takes 15–20 min. Sterilize the gradient by passing through a 0.45µm
filter. Add 5mL of FCS, mix and prepare 2.5 mL aliquots. Store the gradient at
–30°C.We use metrizamide produced by Nygaard, Norway. Metrizamide is also
produced by Sigma, and we have had satisfactory results with Sigma’s
metrizamide.
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 institutional
review board. Extract the lymph nodes from at least three mice and put the lymph
nodes together on the cell strainer submerged in DMEM. Make sure that the
lymph nodes are covered with the medium.
3. Remove the plunger from a syringe and use it to press the lymph nodes through
the 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 15mL conical tube.
Wash cells once with DMEM by centrifuging for 5 min at 300g. Resuspend cells
in 10mL of RPMI-FCS.
5. Thaw the metrizamide gradient and transfer 2.5mL into a sterile 15mL tube.
Overlay cells slowly onto the gradient. It is important not to disturb the gradient.
Spin the gradients for 10 min at 500gat room temperature with no break on the
centrifuge.
6. Collect cells from the interface using a 5mL pipet and wash them once with
RPMI-FCS. Resuspend cells in 1 mL of RPMI-FCS.
7. Count cells on the hemocytometer at magnification 400×. DCs may be clearly iden-
tified by their distinct morphology. This DC enriched fraction can now be used in
DC from Mouse Lymph Nodes 5
morphological or functional studies (seeNotes 1and2). To confirm the purity of
the samples, cells can be analyzed by flow cytometry (seeSubheading 3.3.).
3.2. Purification of DC by Panning
If a higher purity of DCs is desired, DCs can be further enriched by panning
(seeNote 3).
1. Prepare a 6-well plate for panning by coating separate wells with either anti-
mouse immunoglobulin or anti-rat immunoglobulin. We usually use goat anti-
mouse immunoglobulin and goat anti-rat immunoglobulin from Sigma, St. Louis,
MO. Add 3mL of each antibody at a concentration of 1 mg/mL in phosphate-
buffered saline (PBS) to separate wells.
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 (seeNote 4).
3. Wash the DC fraction derived by density gradient centrifugation (seeSubhead-
ing 3.1.) once in PBS and resuspend in 100µL of hybridoma supernatants of
anti-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) (seeNote 5).
4. After a 25-min incubation on ice, wash cells twice and resuspend in 3mL of PBS
containing 0.1% mouse serum.
5. Transfer the cell suspension to a well coated with anti-rat immunoglobulin. Incu-
bate cells on the plates for 60–90 min at 4°C.
6. Harvest the nonadherent DC enriched fraction using a 5mL pipete. Gently wash
the well with ice cold PBS to remove any partially adherent cells and add these to
the nonadherent fraction (seeNote 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 with
anti-mouse immunoglobulin. Incubate for another 60-90 min and then collect
cells as described previously. Cells can now be resuspended in RPMI-FCS and
used for further study (seeNote 5).
3.3. Analysis of DC Purity by Flow Cytometry
We routinely use double labeling with anti-CD11c (N418) antibody and anti-
B7-2 (CD86) antibody to identify DC as CD11c+ CD86+ cells on the flow
cytometer.
1. Transfer 100µL of the purified DC cell suspension into two tubes for flow
cytometry 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 5µL of
isotype control antibodies into the “control” tube.
4. Incubate on ice for 25 min, then wash twice with 2mL of cold PBS and analyze
on the flow cytometer (seeNote 6).
6 Gabrilovich
4. Notes
1. Generally, one can expect to isolate around 5–10×103DCs from one lymph node. For
functional 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 (cid:96)-mercaptoethanol is added to the culture
medium while growing mouse cells. However, (cid:96)-mercaptoethanol should be
added 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 to
use highly purified lymph node DCs, we suggest using the panning technique as
opposed to complement-dependent cytotoxicity. The latter method usually results
in the nonspecific loss of some DC. If the investigator has access to a flow cell
sorter, 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 rat
Ig2 . In the first panning step, T cells and macrophages are eliminated using anti-
b
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 to
plastic 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 flow
cytometer (Becton Dickinson, Mountain View, CA). It is important to note that
there is no single marker that would allow for detection of 100% of lymph node
DCs. 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 of
other 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. Blood90, 3245–3287.
DC from Mouse Lymph Nodes 7
Fig. 1. Flow cytometry demonstrates the purity of DC isolated from lymph nodes
following density gradient centrifugation.
4. Knight, S. C., Farrant, J., Bryant, A., et al. (1986) Non-adherent, low density cells
from human peripheral bloodcontain dendritic cells and monocytes, both with
veiled morphology. Immunology57, 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. Methods184, 81–85.
6. Young, J. W. and Steinman, R. M. (1988) Accessory cell requirements for the
MLR and polyclonal mitogens, as studied with a new technique for enriching
blood dendritic cells. Cell Immunol111, 167–171.
DC from Mouse Spleen 9
2
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 of
the organ means that useful numbers of DC can still be purified. In recent years
the ability to grow cells with the phenotypic and functional properties of DC
from bone marrow progenitors has opened new avenues of research. However,
the relationship of cells grown in this way to DC populations in vivo is
unknown 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 cell-
surface (cid:95)(cid:95) homodimer of the CD8 molecule. The freshly isolated CD8(cid:95)(cid:95)+
population is DEC-205+, CD24+, CD11b-, 33D1-, CD4-, whereas the CD8(cid:95)(cid:95)-
subset is DEC205-, CD24-, CD11b+, 33D1+, CD4-. Both subsets express
CD11c, and this marker appears to be expressed selectively on DC and in the
mouse can be used as a pan-DC marker. The CD8(cid:95)(cid:95)+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(cid:95)(cid:95)-population is probably localized
predominately in the marginal zone, between the red and white pulp, but mobi-
lizes into the T-cell areas in response to lipopolysaccharide (LPS) administra-
tion (5). This marginal zone DC population has a higher phagocytic activity
and turnover rate than the interdigitaing cells (6). The CD8(cid:95)(cid:95)+and CD8(cid:95)(cid:95)-
populations may be cells of lymphoid and myeloid lineages, respectively. They
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
9
10 Stagg et al.
can both activate resting T cells, but may stimulate different types of responses.
The CD8(cid:95)(cid:95)+ population has been reported to drive preferentially Th1
responses, whereas presentation of antigen by the CD8(cid:95)(cid:95)-may favor Th2
responses (7). CD8(cid:95)(cid:95)+ 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 further
heterogeneity with the description of a third, CD4+, spleen DC subset (9).
There are many published protocols for isolating mouse spleen cells and in
choosing among these methods two factors should be borne in mind. First,
different methods may favor the recovery of particular DC subsets at the
expense 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 the
investigator’s advantage in the purification of particular subsets. Second, DC
may be altered phenotypically or functionally by the isolation process itself.
This modulation occurs in methods in which DC are cultured for prolonged
periods, because in vitro culture is sufficient to induce DC maturation. Changes
in properties of DC may also occur during positive selection with monoclonal
antibodies 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 mouse
spleen DC that involves overnight culture and separation on hypertonic
metrizamide gradients and provide a suggested protocol for the further purifi-
cation of these cells. We also describe an alternative method for spleen DC that
avoids the need for culture and discuss how the choice of method for initial
preparation of a spleen cell suspension can be used to influence the recovery of
particular DC subsets.
2. Materials
1. Specific pathogen free mice. The commonly used strains in our laboratory are
BALB/c, CBA, and C3H. We have used mice of either sex, and they are usually
aged 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)
(seeNote 1).
6. HEPES-buffered RPMI-1640 (Sigma, cat. no. R-5886)
7. Metal cell strainers.
8. 60 mm Petri dishes (Nunc or Sterilin).
DC from Mouse Spleen 11
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 glass
equivalents.
12. Small filters for sterilization (GelmanSciences; cat. no. 6224184 [0.45mm] or
cat. 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 (seeNotes 2 and 3).
To prepare collagenase stock:
a. Dissolve 500 mg of collagenase D in 50 mL serum-free HEPES buffered
RPMI-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 dH 0 (10 mg/mL).
2
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) (seeNote 4).
17. Cell scrapers (Falcon; cat. no. 3085).
18. Analytical grade metrizamide (Nycomed; cat. no.22.20.10) (seeNote 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 (seeNote 6).
21. Heat-inactivated normal mouse serum.
22. Monoclonal antibodies and immunomagnetic microbeads (seeNote 7). These include:
a. “Fc-Block” (PharMingen; cat. no. 01241A/D) (seeNote 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 some
applications the same antibody labeled with an alternative fluorochrome (e.g.,
phycoerythrin) may also be required.
12 Stagg et al.
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) (seeNote 9)
24. MiniMACS columns (Type MS+/RS+for miniMACS or varioMACS and/or Type
LS+/VS+ for varioMACS, Miltenyi) (seeNote 9).
3. Methods
3.1. Preparation of Single Cell Suspensions from Spleens
This section describes removal of the mouse spleen and presents three dif-
ferent methods for producing a single-cell suspension from the organ. The way
in which the choice of methods 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 through
the skin just below the ribcage and visualize the spleen.
5. Using a second, smaller, set of instruments, remove the spleen, trimming away
any fatty tissue.
6. Place spleen into complete medium at room temperature (seeNote 10). Spleens
from multiple animals can be pooled.
3.1.2. Preparation of a Single-Cell Suspension using a Metal Sieve
This has been our routine method for many years. It avoids the use of pro-
teolytic enzymes, gives good recovery of DC numbers, and, in conjunction
with overnight culture and metrizamide separation, yields a mixture of CD8(cid:95)(cid:95)+
and CD8(cid:95)(cid:95)- DC (seeFig. 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 milli-
liters of fresh medium.
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 mul-
tiple spleens.)
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 cell
suspension.
7. Top-up tube with complete medium to appropriate volume (see below).
