Abstract
In vitro Th17 (α, β T helper cell which produce IL-17A, IL-17F and IL-22) differentiation has been routinely used for functional T cells studies. Here we describe a method for Th17 cell differentiation.
Keywords: Th17, IL-17, FACS
Background
T cells are critical to mediate host defense against bacteria, viruses and fungi as well as commensal (Kumar et al., 2016). T cells can be further subdivided into T helper (Th1), Th2 and Th17 subsets based on their ability to generate specific cytokines. Naive T cells can be differentiated into specific T cell subsets in in vitro culture in response to specific cytokine stimulation. In vitro generated Th1, Th2 and Th17 cells have helped us to understand the molecular mechanism of their differentiation and their effector functions. Here, we have described a basic protocol for Th17 cell generation.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
FACS Diva or Flow Jo software can be used to analyze data. Plot a linear FSC versus SSC dot plot and create a gate (P1) to select all cells. Using P1 population plot a linear CD3 versus CD4 dot plot. Majority of differentiated cells will be positive for both CD3 and CD4. CD3+CD4+ double positive population (gate P2) will be analyzed for intracellular IL-17 and IL-22 staining.
Recipes
Acknowledgments
The authors would like to acknowledge support from the following PHS grants: P50HL084932, 5R01HL061271, and R37HL079142 to J.K.K. P.K is supported from Children’s Hospital of Pittsburgh Research Advisory Committee Grant from Children’s Hospital of Pittsburgh of the UPMC Health System.
References
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