Permeabilization buffer 10

The eBioscience™ Cell Stimulation Cocktail (lot no.: 2046941) was purchased from Thermo Fisher Scientific Inc. (USA). FITC-conjugated anti-human CD4 (lot no.: B262223), PerCP-Cy5.5-conjugated anti-human CD3 (lot no.: B560835), Brilliant Violet 510-conjugated anti-human IFN-γ (lot no.: B323449), PE-conjugated anti-human IL-17A (lot no.: B315520), APC anti-human FOXP3 (lot no.: 1995356), PE-conjugated anti-human CD25 (lot no.: 2173867), Brilliant Violet 421-conjugated anti-human CD274 (lot no.: B320732), PE/Cyanine7-conjugated anti-human CD279 (lot no.: B318888), Fixation/Permeabilization Concentrate (lot no.: 2084746), eBioscience™ Fixation/Perm Diluent (lot no.: 2047346), Permeabilization Buffer 10× (lot no.: 2060497), and FIX&PERM (Fixation Medium A, Permeabilization Medium B) (lot no.: 17159) were purchased from Biolegend (USA).

To evaluate IFNγ production in activated NK, NKT and T cells, in some experiments Brefeldin A was added at 1:1000 an hour after stimulation or 4 h prior to recovery and surface staining (BioLegend, cat# 420601-BL, 1000×). Monensin and Brefeldin A were added together when cells were to be evaluated for both IFNγ production and CD107a surface exposure. Following standard surface and viability staining cells were processed using the eBioscience Intracellular Staining Buffer Set (ThermoFisher, cat# 00-5523). Briefly, following surface staining with or without anti-CD107a-AlexaFluor 488, cells were fixed for 30 min with 1 part Fixation/Permeabilization Concentrate (cat# 00-5123) and 3 parts of Fixation/Permeabilization Diluent (cat# 00-5223), washed twice with 200 μl/well Permeabilization Buffer 10× (cat# 00-8333), diluted 1:10 in double distilled water), and stained with anti-human IFNγ-APC antibody (BioLegend, cat# 502512, at 1:50) in Permeabilization Buffer for 1 h at RT. Cells were washed twice in Permeabilization Buffer, fixed in 2% PFA in 1xPBS for 15 min at RT, washed again with FACS Buffer to remove PFA and analyzed on BD FACSAria II.