Intracellular staining was performed according to our previous methods (35 (link)). Briefly, for detection of IFN-γ, TNF-α and IL-2, cells were incubated for 4 h with PMA (50 ng/ml) and ionomycin (750 ng/ml) in the presence of GolgiStop (BD Bioscience). For IL-17 and IL-22 detection, cells were cultured with rIL-23 (20 ng/ml) for 16 h. GolgiStop was added at the last 4 h of culture. The inhibitors of the mTORC1/PI3K pathway were added in selected experiments. After incubation, cells were collected, stained with fixable viability dye, blocked with FcγR blocker (CD16/32), and stained for specific surface molecules. After surface staining, cells were fixed, permeabilized and stained for intracellular cytokines by using a fixation/permeabilization kit (eBioscience). Samples were processed on an LSRII FACSFortessa (Becton Dickinson, San Jose, CA) and analyzed by using FlowJo software (TreeStar, Ashland, OR).
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Intracellular Cytokine Staining Assay
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Corresponding Organization : The University of Texas Medical Branch at Galveston
Other organizations : Xiangya Hospital Central South University, Central South University
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Variable analysis
independent variables
- Stimulants (PMA and ionomycin) for detection of IFN-γ, TNF-α and IL-2
- RIL-23 for detection of IL-17 and IL-22
- Inhibitors of the mTORC1/PI3K pathway (added in selected experiments)
- Production of IFN-γ, TNF-α, IL-2, IL-17, and IL-22 by cells
- Incubation time (4 h for PMA/ionomycin, 16 h for rIL-23)
- Cell preparation (staining with fixable viability dye, blocking with FcγR blocker, surface staining, fixation, permeabilization, and intracellular staining)
- Flow cytometry analysis (LSRII FACSFortessa, FlowJo software)
- None explicitly mentioned
- None explicitly mentioned
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