Bioscience cytofix cytoperm kit

Single cell suspensions were obtained as previously described (Kashem et al., 2015a (link)). After doublet and live/dead exclusion, cells were gated as follows: dermal γδ T cells, CD45⁺ CD3⁺ TCRγδ^mid; CD4 T cells, CD45⁺ CD3⁺ TCRαβ⁺ CD4⁺ CD8⁻; DETC, CD45⁺ CD3⁺ TCRγδ^high; CD8 T cells, CD45⁺ CD3⁺ TCRαβ⁺ CD4⁻ CD8⁺; innate lymphoid cells, CD45⁺ CD90.2⁺ Lin⁻ (B220, CD11c, CD11b, F4/80) CD3⁻; neutrophils, CD45⁺ CD11b⁺ Gr-1^high; endothelial cells, CD45⁻CD31⁺; Dendritic Cell, CD45⁺MHCII⁺CD11c⁺; Macrophage, CD45⁺MHCII⁺CD64⁺. Intracellular cytokine staining was performed using BD Bioscience Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA). Samples were analyzed on LSRFortessa flow cytometers (Becton Dickinson, Franklin Lakes, NJ). Data were analyzed using FlowJo software (TreeStar, Ashland, OR). For evaluation of cytokine production, cells were stimulated with PMA (50 ng/ml) and ionomycin (1.5 μM; Sigma-Aldrich, St. Louis, MO), supplemented with GolgiStop (BD PharMingen, San Jose, CA) in RPMI 1640 containing penicillin, streptomycin, 10% FBS, glutamine, nonessential amino acids, and 50 μM of β-mercaptoethanol for 4 hours at 37°3 and 5% CO2. For extracellular staining, antibodies were diluted in staining media (3% Calf Serum, 5mM EDTA, 0.04% Azide).