Goat anti mouse igg af488 a 11029

Antibodies used in this study were obtained from the indicated sources: rabbit monoclonal anti-human PKCδ EP1486Y (Abcam) for WB (this antibody does not recognize mouse PKCδ); rabbit polyclonal anti-rat PKCδ C-17 (Santa Cruz Biotechnology) for WB (recognizes both human and mouse PKCδ); anti-human CD3 UCHT1 (BD Biosciences and Santa Cruz Biotechnology) for cell stimulation and immunofluorescence; rabbit polyclonal anti-phospho-PKCδThr505 (Cell Signaling Technology) for WB; mouse monoclonal anti-CD63 clone NKI-C-3 (Oncogene) for WB; mouse monoclonal anti-CD63 clone TA3/18 (Immunostep) for immunofluorescence; and mouse monoclonal anti-γ-tubulin (SIGMA) for immunofluorescence. Fluorochrome-coupled secondary antibodies (goat-anti-mouse IgG AF488 A-11029, goat-anti-rabbit IgG AF488 A-11034, goat-anti-mouse IgG AF546 A-11030, goat-anti-mouse IgG AF647 A-21236) for immunofluorescence were from ThermoFisher. All horseradish peroxidase (HRP)-coupled secondary Abs (goat anti-mouse IgG-HRP, sc-2005 and goat anti-rabbit IgG-HRP, sc-2004) were obtained from Santa Cruz Biotechnology. Cell tracker blue (CMAC) and phalloidin were from ThermoFisher. Annexin V-PE was from Immunostep. Carbachol (CCH) and staphylococcal enterotoxin E (SEE) were from SIGMA and Toxin Technology, Inc (USA), respectively. Blocking antibody directed against CD95 (Fas), clone DX2, was from BDBiosciences.

Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood using Lymphocyte Separation Medium (Capricorn, Ebsdorfergrund, Germany). Mouse BCH clones were cultured in RPMI-1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine (all Capricorn) and 10% FCS (Merck Millipore, Berlin, Germany). PBMC were washed twice with PBS + 1% FCS and 1 ×10⁶ cells per sample were subsequently stained with Dsg3-AF647 together with mouse anti-human CD19-PerCP-Cy5.5 (HIB19), mouse anti-human CD27-PE (M-T271), mouse anti-human CD38-FITC (HIT2) and the respective isotype controls (all BD Biosciences, Heidelberg, Germany) or with goat anti-mouse IgG-AF488 (A-11029; Thermo Fisher Scientific, Waltham, MA, USA) for mouse BCH clones. After incubation for 20 min at 4°C cells were washed twice with PBS + 1% FCS and a minimum of 2.5 ×10⁵ PBMC or 0.5 ×10⁵ BCH per sample were acquired on a FACS Calibur (BD Biosciences). In a subset of experiments, sorting of cells was performed using FACS Aria III (BD Biosciences). Data analysis was performed using FlowJo 7.6 (TreeStar Inc., Ashland, USA).