F ab 2 rabbit anti mouse anti igg h l

For measurements of intracellular free calcium concentration ([Ca²⁺]_i), RBC-depleted single-cell suspended splenocytes were simultaneously stained with CD8-PE (53–6.7; BD Biosciences), CD4-APC (GK1.5; BioLegend) or B220-PE (RA3–6B2; BioLegend) and loaded with Indo-1 acetoxymethyl (Indo1-AM; Molecular Probes), as described previously (8 (link)). For analysis of ([Ca²⁺]_i), cells were suspended at 10×10⁶ cells/ml in warm IMDM + 2% FBS in a 500ul volume. Cells were acquired for 30 seconds to establish a baseline, and then stimulated with 5μg/ml of F(ab’)₂ rabbit anti-mouse anti-IgG (H&L; Invitrogen) +/− indicated doses of Idelalisib (LC Laboratories) and acquired for 3 minutes. For CD4 and CD8 T cells, cells were acquired for 30 seconds to establish a baseline, and then 10μg/ml of anti-CD3-biotin (145–2C11; BD Biosciences) +/− indicated doses of Idelalisib was added, 60 seconds later 20μg/ml streptavidin (Sigma-Aldrich) was added and acquired for 3 minutes. Mean relative ([Ca²⁺]_i) was monitored over time using an LSR Fortessa X-20 (BD) with analysis using FlowJo software (Tree Star).

RBC-depleted single-cell suspended splenocytes were suspended at 10×10⁶ cells/ml in serum free IMDM, +/− indicated doses of Idelalisib (LC Laboratories) (as indicated in Figure 4) and rested for 1 hour at 37°C. Cells are then washed 2X in serum free IMDM, and stimulated with 5μg/ml of F(ab’)₂ rabbit anti-mouse anti-IgG (H&L; Invitrogen) or 10μg/ml anti-CD3-biotin (145–2C11; BD Biosciences) + 20μg/ml streptavidin (Sigma-Aldrich) for 2 minutes. Signaling was stopped by addition of 20% PFA to a final concentration of 2%, incubated at 37°C for 15 minutes and resuspended in 100% ice-cold MeOH (directly from −80°C). Cells were then placed on ice for 30 minutes and placed at −20°C for storage. For analysis, cells were stained with B220-BV786 (RA3–6B2; BD Biosciences), CD4-BV711 (GK1.5; BioLegend), CD8-BV421 (53–6.7; BioLegend) and/or pAKT-Alexa Fluor 647 (pS373; M89–61; BD Biosciences), pPLCγ-PE (pY759; K86–689.37; BD Biosciences), pBTK-BV421 (pY180; N35–86; BD Biosciences) pSyk/Zap70-PE (pY352/pY319; BD Biosciences) at room temperature for 1 hour. Cells were washed 3 times and samples were acquired in triplicate on an LSR Fortessa X-20 (BD) with analysis using FlowJo software (Tree Star).