Intracellular cytokine staining for flow cytometry (mouse)

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).

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 0.2&mu;m filter; store at 4&deg;
 * stimulation solution
 * 5mL complete DMEM
 * 2.5&mu;L of 200µg/ml PMA (phorbol 12-myristate-13-acetate)
 * 1.35&mu;L of 10mM ionomycin
 * 20&mu;L 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. 100&mu;L; 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&deg;.
 * 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.
 * 10) *Note: for steps 10 through 13, either use all of BD reagents or all of eBioscience reagents. Do not mix-and-match.
 * 11) 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&deg;.
 * 12) Wash once with 200µl either BD Perm/Wash buffer OR 1X eBioscience permeabilization buffer. Spin as in step 10.
 * 13) 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.
 * 14) Wash twice with BD Perm/Wash OR eBioscience permeabilization buffer, spinning as in step 10.
 * 15) Resuspend cells in 100-200µl FACS buffer and transfer to Falcon round-bottom tubes for acquisition on a flow cytometer.