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Flow Cytometry
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.
|
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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