Flow Cytometry Technical Resources

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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

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