Flow Cytometry Cell Stimulation Optimization (for Intracellular Cytokine Staining)

The table below is a handy reference of different stimulants and incubation time vis-à-vis the target proteins.

  • In the case of staining for secreted cytokines, add brefeldin A or monensin during the incubation period at the concentration recommended by the manufacturer.
  • Set aside some unstimulated aliquots for the unstained control and stained baseline controls.

In Vitro Cell Stimulation Reference Table

Target cytokine/phosphoprotein Target cells Stimulant Duration Surface marker
IL-2 PBMCs PMA (50ng/ml) 4-6 hours CD3
IL-3 T-cells PMA(50ng/ml) + ionomycin (1µg(ml) 4-6 hours CD4
IL-4 PBMCs PMA(50ng/ml) + ionomycin (1µg(ml) 4-6 hours CD4
IL-6 PBMCs LPS (100ng/ml) 4-6 hours CD14
IL-10 PBMCs LPS (100ng/ml) 18-24 hours CD14
GM-CSF /IFNγ/TNFα/TNFβ PBMCs PMA(50ng/ml) + ionomycin (1µg(ml) 4-6 hours CD3
pStat5 PBMCs GM-CSF (20ng/ml) + IL3 (20ng/ml) 15 min. CD123, CD116
pStat3 PBMCs G-CSF (20ng/ml) + IL6 (20ng/ml) 15 min. CD126, CD114
pERK PBMCs IL3 (20ng/ml) + IL6 (20ng/ml) + FLT3L (20ng/ml) 15 min. CD123, CD126, CD135


Keywords: FACS in vitro cell stimulation, step wise protocol