Download Culture and Identification of Human Monocyte Derived Dendritic Cells

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Technical Protocol
October 2013
Culture and Identification of Human
Monocyte Derived Dendritic Cells
Introduction
Dendritic cells (DCs) are professional antigen presenting
cells (APCs) that play a critical role in regulation of adaptive
immune responses. They are also referred to as a missing link
between innate and adaptive immune responses. DCs perform
the important role of capturing and processing antigen and
presenting it to naive T-helper cells to initiate an immune
response.
Distinct subsets of DCs have been described and they show
great heterogeneity. Their phenotype changes according to
their stage of differentiation or according to their response
and interaction with cytokines and/or growth factors in their
microenvironment. The phenotypes described in the literature
are highlighted in the tables shown in Appendix 1.
DCs present a scientific challenge because there is presently
no single monoclonal antibody that recognizes all dendritic
cells. This protocol focuses primarily on blood dendritic cells.
The two types of DCs identified in large numbers in blood, the
myeloid DCs (CD11c+/CD123–) and plasmacytoid DCs (CD123+/
CD11c–). These are immature, resting DCs in the peripheral
blood which lack the expression of known lineage markers
such as CD3, CD19, CD56, CD16, and CD14 bright. Cultivation
of peripheral blood mononuclear cells (PBMCs) in the presence
of GM-CSF, IL-4, and TNF has been shown to induce the
differentiation of monocyte-derived DCs (MDCs) in vitro. The
phenotypic characteristics observed in cultured dendritic cells
are different from cultured PBMCs. The addition of growth
factors and cytokines induce cultured MDCs to increase in
CD40, CD80, CD83, CD86, and HLA-DR expression while
decreasing CD14 and CD1a.
Using the recommended reagents affords researchers the
ability to optimize the incubation time to best suit their
needs. Under these culture conditions, one is able to analyze
a greater number of cells than in peripheral blood or tissue.
We recommend that the cells be cultured for a minimum of
three days to start observing the characteristic phenotype. A
panel of monoclonal antibodies is also suggested: they are
useful to perform immunophenotypic analysis on MDCs. These
antibodies do not indicate or demonstrate any functional
characteristics of the cells, but do indicate the expression of the
molecules associated with dendritic cell differentiation.
Culture and Identification of Human Monocyte Derived Dendritic Cells
Cell Culture Protocol
Reagents Required
Full Name
Short Name
Cat. No.
BD Recombinant Human IL-4
IL-4
554605
BD Recombinant Human GM-CSF
GM-CSF
550068
Lipopolysaccharide
LPS
Sigma L-8274
RPMI-1640 w/o L-glutamine
Incomplete RPMI
Sigma R0883
RPMI-1640 w/10% fetal bovine serum
Complete RPMI
T-75 Tissue Culture Flask
T-75
353110
Ficoll-Hypaque (Amersham Pharmacia Biotech AB)
BD Vacutainer® blood collection tubes
17-0840-02
tubes
366480
• Dilution Buffer: IMDM with 25 mM HEPES buffer with sodium bicarbonate
• Culture medium: IMDM, 10% FBS, 1% glutamine, 1% Pen-strep media
• PBS
Additional Materials Suggested
Devices
2-color capable flow cytometer (with 488-nm laser )
FL1-FITC (520 nm), FL2-PE (576 nm), with FL1 detection
through a 515–545-nm bandpass filter and FL2 detection
through a 561–606-nm bandpass filter.
Centrifuge
Beckman TJ-6 or similar with swinging bucket rotor capable of
centrifugation of at least 2,000 RPM.
Fluid Handling
Polypropylene tubes: 12 × 75-mm and 17 × 120-mm (15 mL)
Graduated conical centrifuge tubes: 50 mL
Micropipettes
Capable of dispensing 20-μL, 100-μL and 500-μL volumes
Biologics
Recombinant human TNF (Cat. No. 554618)
Vortex mixer
Aspirator
Procedural Notes
• Use of freshly prepared PBMCs is recommended. If frozen PBMCs must be used, conditions for post-thaw cell recovery should be
optimized to ensure appropriate cellular responsiveness to stimuli.
• The activity of our recombinants is lot-specific, so some optimization might be required.
• Most molecules show good expression by the third or forth day of culture. Optimal expression is usually seen around the fifth
and sixth day of culture. Cell viability might decline after the seventh day.
• DCs will be sticky on the plates and need to be removed carefully to collect as many as possible and increase cell numbers.
Cell Separation Procedure
1. Collect blood in a sodium heparin tube.
5. Centrifuge at room temperature, 400g, for 20 minutes.
2. Dilute blood 1:2 in PBS.
3. Add 5 mL of Ficoll-Hypaque to a labeled 15-ml conical
centrifuge tube.
6. Carefully remove the top layer and discard. Transfer the cells
at the interface of the solution into a clean 15-mL conical
tube.
4. Add 10 mL of diluted peripheral blood to the top of the
5-mL Ficoll-Hypaque (overlay).
7. Wash twice with dilution buffer. Centrifuge at 200g,
for 10 minutes.
8. Suspend the PBMCs in IMDM culture medium at
4 × 106 cells per mL.
Cell Culture Procedure
1. Add 8 mL of the cell suspension into a T-25 culture flask and
incubate at 37°C, 7% CO2 for 2–3 hours.
4. Incubate at 37°C, 7% CO2, at 100% humidity for
4 to 7 days.
2. Gently shake and discard non-adherent cells by washing
twice with pre-warmed culture medium.
5. Add recombinant TNF to a final concentration of 20 ng/mL
on the fifth day of incubation.
3. Add 10 mL of culture medium with a final concentration of
20 ng/mL and GM-CSF 20 ng/mL IL-4.
6. Harvest the cells as cultured human MDCs.
Page 2
Culture and Identification of Human Monocyte Derived Dendritic Cells
Cell Immunophenotyping Protocol
Reagents Required
Full Name
Short Name
Cat. No.
CD1a FITC clone HI149
CD1a FITC
555806
CD40 PE clone 5C3
CD40 PE
555589
HLA-DR PE Clone G46-6
HLA-DR PE
555812
CD80 FITC clone L307.4
CD80 FITC
557226
CD86 FITC clone 2331
CD86 FITC
555657
CD83 PE clone HB15e
CD83 PE
556855
CD14 PE clone M5E2
CD14 PE
555398
Isotype control PE-IgG1
IgG1 PE control
555749
Isotype control FITC-IgG1
IgG1 FITC control
555748
Isotype control PE-IgG2a
IgG2a PE control
555574
• Wash Buffer: PBS + 1% FBS +1% sodium azide
Procedural Notes
• Each cell surface molecule will vary in expression with culture time and donor.
• We recommend that each investigator titrate the antibody to obtain optimal results.
• For analysis, set the cytometer with the lymphocytes at the very low left corner in as tightly as possible to see the DCs, since the
DCs are observed as large cells by FSC vs SSC.
• Although cultured dendritic cells have a different light scatter than that of whole blood or PBMCs, the instrument settings used
for whole blood or PBMC analysis are usually adequate. DCs are larger in size and have greater granularity than lymphocytes and
monocytes.
Staining Procedure
1. Adjust the cell concentration to 0.5–1 × 106 cells/mL.
2. Add 100 μL of cell suspension to each 12 × 75-mm tube.
3. The following controls should be included:
– Cells with PBS
– Cells with isotype control
4. Add the monoclonal antibodies into the tubes at the
predetermined concentration. Mix well.
5. Incubate in the dark at room temperature for 20–30
minutes.
6. Add 2 mL of wash buffer. Mix well and centrifuge for 5
minutes at 200g.
7. Aspirate the supernatant, resuspend the cells in 0.5 mL of
wash buffer, and vortex.
8. Analyze by flow cytometry.
Page 3
Culture and Identification of Human Monocyte Derived Dendritic Cells
Page 4
Appendix 1
Different DC subtypes and their salient surface markers from literature
DC subset
DC type
Key surface markers
DC subset
DC type
Key surface markers
Plasmacytoid DCs
Lymphoid resident DCs
B220 DC
SIGN++CD207–CD103–
CD205–CD11b–EPCAM–
CD8a+++
CD11b DCs
Migratory DCs
B220–DC–SIGNndCD207–
CD103+/–
CD205+EPCAM–CD8a–
CD11b+
CD8a+ DCs
Lymphoid resident DCs
B220-DC–SIGN–CD11b+
CD207+/CD103lowCD205+
EPCAM–CD8a–
CD103+ DCs • lung
Migratory DCs
B220–DC–SIGN–
CD207+CD103+CD205++
EPCAM+/–CD8aa–
CD11b–
CD4+ DCs
Lymphoid resident DCs
B220–DC–SIGN–
CD11b+CD207–CD103–
CD205–EPCAM–CD8a–
CD103+ DCs •
intestine
DN DCs
Lymphoid resident DCs
B220–DC–SIGN–
CD11b+CD207–CD103–
CD205–EPCAM–CD8aa–
+
–
+
DC subset
B220–DC–SIGN–CD207–
CD103+CD205–
EPCAM–CD8a–CD11b+
DC type
Key surface markers
Langerhans cells
B220–DC–SIGN–
CD207++CD103–
CD205++EPCAM+
CD8a–CD11b+
Monocyte-derived
DCs
B220–DC–SIGN+CD207–
CD103–CD11b+CD205–
EPCAM–CD8a–
References
Immunophenotyping
Belz GT, Nutt SL. Transcriptional programming of the dendritic cell network. Nat Rev Immunol. 2012;12:101-113.
Yan de Laar L, Coffer PJ, and Wolfman AM. Regulation of dendritic cell development by GM-CSF: molecular control and implications for immune
homeostasis and therapy. Blood. 2012;12,119:3383-3393.
Other Markers
Azuma M, Ito D, Yagida H, et al. B70 antigen is a second ligand for CTLA-4 and CD28. Nature, 1993;366:76-79.
Barclay AN, Brown MH, Law SKA, et al, eds. The Leucocyte Antigen Facts Book, second edition. MHC Class II Section. San Diego, California:
Academic Press, Inc. 1997;567.
Engel P, Gribben JG, Freeman GJ, et al. The B7-2 (B70) costimulatory molecule expressed by monocytes and activated B lymphocyte is the CD86
differentiation antigen. Blood. 1994;84:1402-1407.
Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood.1997;90:3245-3287.
Kishimoto T, Kikutani H, von dem Borne AEG, et al, eds. Leucocyte Typing VI: White Cell Differentiation Antigens. London: Garland Press;1998.
Nunez R, Filgueira L. Flow cytometric assessment of the reactivity of a panel of monoclonal antibodies (mAb) against two populations of human
dendritic cells (DC). BMC Immunol. 2001;2:6.
Schwartz RH. Costimulation of T lymphocytes: the role of CD28, CTLA-4, and B7/BB1 in interleukin-2 production and immunotherapy. Cell.
1992;71:1065-1068.
Wright SD, Ramos RA, Tobias PS, Ulevitch RJ, Mathison JC. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein.
Science. 1990;249:1431-1433.
Zhou L-J, Tedder TF. Human blood dendritic cells selectively express CD83, a member of the immunoglobulin superfamily.
J Immunol.1995;154:3821-3835.
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