Simultaneous detection of murine antigen-specific intracellular cytokines and CD107a/CD107b by flow cytometry

doi:10.1038/nprot.2007.438

Method Article

Simultaneous detection of murine antigen-specific intracellular cytokines and CD107a/CD107b by flow cytometry

https://doi.org/10.1038/nprot.2007.438

This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.

Version 1

posted

You are reading this latest protocol version

Biological techniques

flow cytometry

intracellular cytokine

degranulation

antigen-specific

Assays to detect cytokine production and degranulation are useful measures of T cell function. Multiparametric flow cytometry makes it possible to precisely phenotype and simultaneously detect these intermediate stages of T cell activation.^1,2 The degranulation of cytotoxic cells, an indicator of cytotoxic potential, results in the transient surface expression of CD107a and CD107b (lysosomal associated membrane proteins).³ A protocol for the ex vivo assessment of antigen-specific cytokine production by mouse CD4+ and CD8+ T lymphocytes, and the simultaneous presence of CD107a and CD107b, by polychromatic flow cytometry is presented here. In this protocol, C57BL/6 or Balb/c mice were immunized with a live-attenuated strain of Listeria monocytogenes (actA-Lm). Splenocytes were restimulated in vitro with peptide from listeriolysin (LLO91-99 for CD8+ (Balb/c), LLO190-201 for CD4+ (C57BL/6)) or PMA plus ionomycin. A typical surface staining cocktail consisted of CD4-APC, and CD8-PerCP-CY5.5 and a typical intracellular staining cocktail consisted of IFN-γ-PE and TNF-α-PE-Cy7. To obtain optimal surface staining for CD107a and CD107b (FITC conjugates) the antibodies were added for the entire activation period. Staining during activation allows access of the antibody to transiently expressed CD107a and CD107b antigens.

15 ml polypropylene centrifuge tube (BD Falcon™, Cat# 352196)
Complete culture media - RPMI 1640 with 10% FBS, 1% L-glutamine and Penicillin-streptomycin (cRPMI, sterile filtered)
Cell culture dish (60mm x 15mm, polystyrene, Corning, Cat# 430166)
Cell dissociation wire screen (mesh size 50, Sigma, Cat# S0895-5EA)
50 ml polypropylene centrifuge tube (BD Falcon, Cat# 352070)
Dulbecco’s phosphate buffered saline (PBS) without Ca²⁺ and Mg²⁺ (Hyclone, Cat# SH30028.03)
PharmLyse 10X, red blood cell lysing buffer (BD Biosciences Cat# 555899)
Cell strainer (40µm nylon, BD Falcon Cat# 352340)
Phorbol 12-myristate 13-acetate (PMA, Sigma, Cat# P-8139)
Ionomycin (Sigma, Cat# I-0634)
Antigen-specific peptides
FITC-anti-mouse CD107a (BD Biosciences, Cat# 553793)
FITC-anti-mouse CD107b (BD Biosciences, Cat# 558758)
Fc Block: rat anti-mouse CD16/CD32 (BD Biosciences, Cat# 553142)
FACS wash buffer – PBS without Ca²⁺ and Mg²⁺ containing 1% bovine serum albumin (Sigma, Cat# A-7906) + 0.1% Sodium Azide (NaN3) (Sigma, Cat# S-80-32), Sterile filtered
APC-anti-mouse CD4 (BD Biosciences, Cat# 553051)
PerCPCy5.5-anti-mouse CD8a (BD Biosciences, Cat# 551162)
PE-anti mouse IFN-γ (BD Biosciences, Cat# 554412)
PE-Cy7-anti-mouse TNF-α (BD Biosciences, Cat# 557644)
Perm Wash Buffer 10X solution (BD Biosciences, Cat# 2091KZ)
Round bottom 96-well cell culture plate, sterile (BD Falcon, Cat# 353077)
BD GolgiPlug™ (Brefeldin A, BD Biosciences, Cat# 555029)
BD GolgiStop™ (monensin, BD Biosciences, Cat# 554724)
Cytofix/Cytoperm 1X solution (BD Biosciences, Cat# 2092KZ)

BD LSRII or equivalent multicolor flow cytometer

A. Cell Preparation

Sacrifice mouse and harvest spleen.
Place spleen in a 15ml polypropylene tube containing 5 ml of cRPMI. Keep on ice until ready to use.
Pour the spleen and media into a cell culture dish containing a wire mesh screen.
Prepare a single cell suspension of the splenocytes by gently pushing the spleen tissue through the wire screen with the rubber plunger end of a 5 ml syringe. Use as little force as possible. Most connective tissue should remain on screen.
Transfer cell suspension from the culture dish into a 15 ml polypropylene tube.
Place the 15 ml tube, containing the cell suspension, upright on ice for 3-5 minutes (until clumps settle to bottom).
Transfer the cell suspension to a 50 ml tube with a pipet. Leave the clumps behind.
Dilute the cell suspension to 50 ml by adding 1X PBS.
Centrifuge cells @ 400 x g for 5 minutes at room temperature. Discard supernatant.
Add 3–5 ml of 1X PharmLyse to cell pellet and mix by gently pipetting up and down.
Let cell suspension stand at room temperature for 7 minutes.
Add 10 ml of cRPMI to the cells and mix by gently pipetting up and down.
Pass cell suspension through a 40 µm cell strainer into a fresh 50 ml tube.
Dilute the cell suspension to 50 ml by adding 1X PBS.
Centrifuge cells @ 400 x g for 5 minutes at room temperature. Discard supernatant.
Wash with 1X PBS two more times (repeat steps 14 and 15).
Resuspend cells in 5 ml of cRPMI.
Count the splenocytes and bring the sample to a concentration of 10x10⁶ cells/ml in cRPMI.

B. Preparation of Activation and Staining Reagents

PMA: Prepare a stock solution of 1 mg/ml in DMSO, aliquot and store at -20°C. This solution is stable at -20°C for at least six months. On day of experiment, serially dilute an aliquot of PMA stock from freezer 1:1000 in PBS to make 1 μg/ml concentration.
Ionomycin: Prepare a stock solution of 1 mg/ml in DMSO, aliquot and store at -20°C. This solution is stable at -20°C for at least six months. On day of experiment, dilute an aliquot of frozen ionomycin stock 1:10 in PBS to make 100 μg/ml concentration.
Peptide: Peptides representing optimal antigenic epitopes are superior to whole proteins for in vitro restimulation. Prepare a sterile peptide stock solution of 2 mM in DMSO, aliquot and store at -80°C. This solution is stable at -80°C for at least six months. On day of experiment, serially dilute the frozen peptide aliquot 1:1000 in cRPMI to make 2 µM concentration.
CD107a/CD107b-FITC Cocktail: on day of experiment combine 0.1 µg CD107a-FITC with 0.5 µg CD107b-FITC in 1X PBS. Prepare enough for all required wells such that 10 µl of the cocktail is added to each well.
Fc Block: Add Fc block to FACS wash buffer at a 1:50 ratio. Make enough to add 100 μl per well (98 μl FACS wash buffer + 2 μl Fc block per well).
Prepare cell surface staining mAbs at optimal (previously determined) concentrations by diluting in FACS wash buffer. Mix all surface mAbs in the same tube so that the total volume added per well is 50 μl.
Prepare intracellular staining mAbs (i.e., cytokines) at optimal (previously determined) concentrations by diluting in Perm Wash buffer. Mix all intracellular staining mAbs in the same tube so that the total volume added per well is 50 μl.

C. Activation and Staining

Prepare working stock of PMA+Ionomycin activating reagent. 100 μl of working stock is needed for every well. To prepare 1 ml, add 2 μl of GolgiPlug, 2 μl of GolgiStop, 40 μl of the diluted PMA (prepared in Step 1 of previous section), and 40 μl of the diluted ionomycin (prepared in Step 2 of previous section) to 916 µl cRPMI. Store on ice until ready to use. Discard leftovers after activation.
Prepare working stock of unstimulated control. 100 μl of working stock is needed for every well. To prepare 1 ml, add 2 μl of GolgiPlug and 2 μl of GolgiStop to 996 µl cRPMI. Store on ice until ready to use. Discard leftovers after activation.
Prepare working stock of peptide activating reagent. 100 μl of working stock is needed for every well. To prepare 1 ml, add 2 μl of GolgiPlug, 2 μl of GolgiStop, and 1 μl of the diluted peptide (prepared in Step 3 of previous section) to 995 µl cRPMI. Store on ice until ready to use. Discard leftovers after activation.
Dispense 100 μl of cell suspension (1x106 cells) into appropriate wells of a 96-well round-bottom cell culture plate. Avoid cell clumps and cells which have settled to the bottom of the tube.
Dispense cells into additional wells (one well per color plus an unstained control) for use as instrument setup and compensation controls. These cells will be stained with a single antibody each (e.g., CD4-FITC, CD4-PE, CD4-PE-Cy7, CD4-PerCP-Cy5.5, or CD4-APC).
Add 100 μl of the working stock of each activator to the appropriate wells.
Add 10 μl of CD107a/CD107b-FITC cocktail (prepared in Step 4 of previous section) to each well as required.
Mix wells by pipetting up and down several times.
Incubate 5-6 hrs at 37°C, 7% CO2.
Optional step – after incubation, the procedure can be interrupted for overnight storage (not to exceed 20 hours) of cells by transferring the plate to 4°C (refrigerator).
Centrifuge the plate at 400 x g for 5 min at 4°C.
Remove supernatant from the wells by quickly “flicking” the plate upside down.
Add 100 μl Fc block (prepared in Step 5 of previous section) to each well and mix by pipetting up and down.
Incubate plate on ice for 20 min.
Add 100 μl of FACS wash buffer to each well.
Centrifuge the plate at 400 x g for 5 min at 4°C.
Remove supernatant by flicking the plate.
Add 50 μl of cell surface staining mAbs (prepared in Step 6 of previous section) to the appropriate wells, mix by pipetting up and down.
Incubate in the dark, on ice, for 30 min.
Add 150 μl of FACS wash buffer to each well.
Centrifuge the plate at 400 x g for 5 min at 4°C.
Remove supernatant by flicking the plate.
Add 200 μl FACS wash buffer to each well, mix by pipetting up and down.
Centrifuge the plate at 400 x g for 5 min at 4°C.
Remove supernatant by flicking the plate.
Add 100 μl Cytofix/Cytoperm to each well and mix by pipetting up and down.
Incubate in the dark, on ice, for 30 min.
Optional - procedure can be interrupted at this point by freezing the cells. Wrap plate in parafilm and foil. Transfer to a -80°C freezer. To continue the procedure, thaw the plate at 37°C for ~20 min.
Add 100 μl of 1X Perm Wash Buffer to each well.
Centrifuge the plate at 500 x g for 5 min. Note that the cells are permeabilized and fixed at this point and require stronger centrifugation to pellet. Be sure to centrifuge at 500 x g for this and all following centrifugation steps
Remove supernatant by flicking the plate.
Add 50 μl of intracellular staining mAb (prepared in Step 7 of previous section) to appropriate wells, mix by pipetting up and down.
Incubate in the dark, on ice, for 30 min.
Add 150 μl Perm Wash buffer to each well.
Centrifuge the plate at 500 x g for 5 min at 4°C.
Remove supernatant by flicking the plate.
Add 200 μl Perm Wash buffer to each well, mix by pipetting up and down.
Centrifuge the plate at 500 x g for 5 min at 4°C.
Remove supernatant by flicking the plate.
Add 200 μl of FACS wash buffer to each well, mix by pipetting up and down.
Cells are ready for acquisition (leave cells in the plate for acquisition on an HTS plate reader, or transfer samples to 12x75 ml tubes for single tube acquisition).
Acquire samples on a BD LSRII or equivalent multicolor flow cytometer. Acquire at least 30,000 CD4+ or CD8+ T cells.

Spleen recovery and preparation – 30-60 minutes

Preparation of activation and staining reagents – 30 minutes

Activation procedure – 1 hour set up plus 5-6 hours incubation

Staining – 2.5-3 hours

FACS analysis – dependent on number of samples and acquisition rates

Betts, M.R. et al. Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J. Immunol. Methods 281, 65-78 (2003).
Suni, M.A. et al. Ex vivo analysis of T-cell function. Curr Opin Immunol 17, 434-440 (2005).
Casazza, J.P. et al. Acquisition of direct antiviral effector functions by CMV-specific CD4+ T lymphocytes with cellular maturation. J. Exp. Med. 203, 2865-2877 (2006).

We would like to thank Laurel Nomura (BD Biosciences) for her work on the initial development of this protocol.

Download PDF

Version 1

posted

You are reading this latest protocol version

Simultaneous detection of murine antigen-specific intracellular cytokines and CD107a/CD107b by flow cytometry

Status:

Version 1

Figures

Introduction

Reagents

Equipment

Procedure

Timing

References

Acknowledgements

Status:

Version 1

Privacy Policy

Terms of Service

Simultaneous detection of murine antigen-specific intracellular cytokines and CD107a/CD107b by flow cytometry

Status:

Version 1

Figures

Introduction

Reagents

Equipment

Procedure

Timing

References

Acknowledgements

Status:

Version 1

Privacy Policy

Terms of Service

Manage Cookie Preferences