Anti pstat1 alexa488

Fresh pre-warmed heparinized whole blood was stimulated in vitro with or without 600 ng/mL consensus sequence IFN-α (IFN-α-con1; InterMune, Brisbane, CA) for 5 min at 37°C. A 20-fold excess of Lyse/Fix buffer (BD Biosciences, San Jose, CA) was added for 10 min at 37°C for lymphocyte fixation and erythrocyte lysis. After centrifugation, cells were permeabilized with Perm Buffer (BD Biosciences) for 20 min on ice, washed twice, and resuspended in Staining Buffer (BD Biosciences).
All samples were stained with anti-CD56-PE (Beckman Coulter, Brea, CA) and anti-CD20-PerCP/Cy5.5 (BD Biosciences) to identify NK cells and B cells, respectively, and with anti-CD3-APC (both from BD Biosciences) to exclude T cells. Cells were additionally stained with anti-pSTAT1-Alexa488, or anti-pSTAT4-Alexa488 (BD Biosciences) for 20 min at room temperature. Samples were analyzed on an LSRII with FacsDiva Version 6.1.3 (BD Biosciences) and FlowJo Version 8.8.2 (Tree Star, Ashland, OR) software.

Thawed PBMCs (1.5×10⁶) were rested in 0.5 ml RPMI 1640 with 10% FBS, 1% penicillin/streptomycin and 2 mM L-glutamine (Mediatech) for 2h. Thereafter, cells were fixed with BD Cytofix buffer for 10 min at 37°C and 5% CO₂, centrifuged, permeabilized with BD Perm buffer III for 20 min on ice, washed twice and resuspended in BD Phosflow Buffer (all from BD Biosciences). Cells were subsequently stained with anti-CD56-PE (Beckman Coulter, Brea, CA), anti-CD3-APC, anti-CD20-PerCP/Cy5.5 and anti-pSTAT1-Alexa488 (all from BD Biosciences) for 20 min at room temperature.