Intracellular cytokine staining for flow cytometry (mouse)

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Overview

Staining for intracellular cytokines, followed by flow cytometry analysis, can provide single-cell information about a cell population that one cannot obtain from surface staining or ELISA (enzyme-linked immunosorbent assay).

Materials

Supplies

• 96-well V-bottom plate
• 12 x 75mm Falcon round-bottom tubes

Reagents

• DMEM-10
  • 1L DMEM (with 4.5g/L glucose, L-glutamine, sodium pyruvate; from Mediatech, catalog# 10-013-CM)
  • 100mL fetal bovine/calf serum
  • 10mL 100x PSG (penicillin G sodium, streptomycin sulfate, L-glutamine; from Gibco, catalog# 10378-016)
  • 10mL HEPES buffer solution (1M)
  • 10mL non-essential amino acids solution (10mM, 100X; Gibco catalog# 11140)
  • 1mL 2-mercaptoethanol (1000X)
    • sterilize using 0.2μm filter; store at 4°
• stimulation solution
  • 5mL complete DMEM
  • 2.5μL of 200µg/ml PMA (phorbol 12-myristate-13-acetate)
  • 1.35μL of 10mM ionomycin
  • 20μL of 10mg/ml brefeldin A
    • chemicals will stick to plastic; make just before use, and discard solution afterwards
• FACS buffer
  • 97mL PBS (phosphate buffered solution)
  • 3% fetal bovine/calf serum (i.e. 3mL)
  • 0.1% sodium azide (i.e. 100μL; optional, especially if you do not plan to store the buffer after use)
• PBS (phosphate buffer solution)
• BD Cytofix/Cytoperm solution (or 4X eBioscience permeabilization solution and eBioscience permeabilization diluent)
• BD Perm/Wash buffer (or 10X eBioscience permeabilization buffer)
• antibodies
  • Fc block (2.4G2)
  • fluorochrome-linked surface markers (e.g. CD3e, CD4, CD8)
  • fluorochrome-linked cytokine antibodies (e.g. IFN-gamma, IL-12)

Equipment

• P200 pipette
• P200 or P300 multichannel pipette (optional)
• flow cytometer

Procedure

1. Dilute single-cell suspensions to 10x10^6 cells/mL in complete DMEM.
2. Add 100µl cells per well (do not forget to make wells for your staining controls).
3. Add 100µl stimulation mix (final concentrations: PMA = 50 ng/ml, ionomycin = 1µg/ml, brefeldin A = 10µg/ml)
4. Incubate 37° for 4 hours.
5. Spin plate at 800 x g, 3 minutes, at 4°.
6. Wash 3 times with cold PBS, spinning as in step 5.
7. Resuspend in 100µl Fc block (recommended dilution: 1:1000 in FACS buffer). Incubate on ice, 10 minutes. Spin.
8. Resuspend in 100µl surface antibody mixture (recommend dilution: 1:200 in FACS buffer). Incubate on ice, 20 minutes in the dark. Spin.
9. Wash once with cold PBS.
   • Note: for steps 10 through 13, either use all of BD reagents or all of eBioscience reagents. Do not mix-and-match.
10. Resuspend in 200µl of either BD Cytofix/CytoPerm solution OR 1X eBioscience permeabilization solution. Incubate on ice, 30 minutes in the dark. Spin 1500 x g, 5 minutes, 4°.
11. Wash once with 200µl either BD Perm/Wash buffer OR 1X eBioscience permeabilization buffer. Spin as in step 10.
12. Resuspend in 100µl cytokine stain (recommended dilution: 1:100 in 1X Perm/Wash OR permeabilisation buffer). Incubate on ice, 30 minutes in the dark. Spin as in step 10.
13. Wash twice with BD Perm/Wash OR eBioscience permeabilization buffer, spinning as in step 10.
14. Resuspend cells in 100-200µl FACS buffer and transfer to Falcon round-bottom tubes for acquisition on a flow cytometer.

Notes

• If you make the FACS buffer fresh every time, there is no need to add sodium azide to the buffer.
• All antibody concentrations here are only recommendations. You should titrate the antibody concentrations for your specific cell populations.
• Non-commercial reagents can also be used for this protocol. See Current Protocols, Unit 6.24 [1].
• As you are permeabilizing the cells through this protocol, the cells are not viable. You cannot sort your cells based on intracellular staining.

References

1. Current Protocols in Immunology, Unit 6.24: Detection of Intracellular Cytokines by Flow Cytometry link (subscription required)

2. eBioscience, Protocol for IC Staining link (scroll to bottom of page)

3. BD Biosciences, Protocols: Immunofluorescent Staining of Intracellular Cytokines for Flow Cytometric Analysis link