Transcription factor fixation permeabilization kit

Colonic lamina propria cells were stained with a Zombie UV fixable stain kit (Biolegend, San Diego, CA, USA). Washed cells were incubated with mouse Fc blocking reagent (Miltenyi Biotec, Auburn, CA, USA), as per the manufacturer’s instructions, before staining with combinations of the following antibodies (or their corresponding isotype controls): CD45 (30-F11), CD3 (145-2C11), CD4 (RM4-5), IL-17A (TC11-18H10.1)/Rat IgG1, κ, IL-10 (JES5-16E3)/Rat IgG2b, κ, RORγt (AFKJS-9)/Rat IgG2a, κ, IL-27 (355025) /Rat IgG2a, and cMAF (sym0F1)/ Rat IgG2b κ. To detect intracellular cytokines, cells were fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences, San Jose, CA, USA). Colonic T cells were stimulated with a cell activation cocktail containing Brefeldin A (Biolegend, San Diego, CA, USA) for 3 h. The Transcription Factor Fixation/Permeabilization Kit (Thermo Fisher Scientific, Grand Island, NY, USA) was used for RORγt and cMAF staining. After staining, a BD LSRFortessa (BD Biosciences, San Jose, CA, USA) cell analyzer was used to acquire fixed cells. Data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Antibodies, and their corresponding isotype controls, were purchased from eBioscience (San Diego, CA, USA), Biolegend (San Diego, CA, USA), BD Pharmingen, or R&D Systems (Minneapolis, MN, USA).