Intracellular cytokine staining for flow cytometry (mouse)

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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).



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


  • 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)


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


  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.


  • 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.


  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