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Protocol for human whole blood intracellular cytokines: assay and staining procedures.

VERY IMPORTANT - blood must be collected into heparinised tubes.

1. Aliquot 0.5ml heparinised whole blood into each of 2 12x75mm snap cap tubes. Add 0.5ml RPMI 1640 w/o additives to each tube.
2. To the first tube add 10µg/ml Brefeldin A. (Brefeldin A is made up at a concentration of 0.5mg/ml in 200 proof EtOh and stored at -20°C. Thus 20µl of the stock solution is added.) Mix contents of tube gently. This tube is the resting cell population.
3. To the second tube add 10µg/ml Brefeldin A, 25ng/ml PMA (PMA is made up at a concentration of 1mg/ml in DMSO and stored at -20°C. Thus dilute stock solution 1/100 in RPMI 1640 w/o additives and add 2.5µl to tube.) and 1µg/ml ionomycin (Ionomycin is made up at a concentration of 1mg/ml in DMSO and stored at -20°C. Thus add 1µl to the tube.) Mix contents of tube gently. This is the activated cell population.
4. Incubate for 4h at 37°C in 7.5% humidified CO2 incubator.
5. At the end of the incubation period mix cells again and aliquot 100µl diluted whole blood into 12x75mm snap cap tubes.
6. Add 5µl of appropriate antibody for phenotyping cells. This antibody could be e.g. CD3- Tri Color(TC), CD4 - TC, CD8- TC, CD45- TC, CD69-TC.
7. Incubate in the dark for 15 mins at RT.
8. Add 100µl reagent A of the Fix&Perm Kit. Mix cells gently and incubate for a further 15mins at RT in the dark.
9. Wash twice with wash medium (PBS+1%BSA, 0.1% NaN3, 1% Fetalclone). The pellet becomes less cohesive on the second wash so use caution so the cells are not lost.
10. To the washed cells add 100µl reagent B of the Fix&Perm Kit and 1-10µl of anti-cytokine antibody OR 5µl of isotype control.
11. Incubate for 20mins at 4°C.
12. Wash twice with wash medium and resuspend pellet in FACS fix.
13. Analyse by gating on SSC and FL-3.

A sample staining protocol is shown below:

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