Annexin 5 pe

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.

Living cells were adhered on PolyPrep slides, fixed with 2 %-PFA and treated with Dapi (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich) for staining the nuclei. Images were obtained with a Leica DMI 4000B Inverted Microscope (Leica Microsistemas, Barcelona, Spain). The percentage of apoptotic events was calculated by acquiring 60 fields from three independent experiments—containing an average number of cells close to 40—and considering the total number of cells. Activation of casapse-3 was measured in non-treated cells with CaspaseGlo^®3/7 systems (Promega). The luminescent signal (relative light units, RLUs), which was directly proportional to caspase activation, was quantified in an Orion Microplate Luminometer with Simplicity software (Berthold Detection Systems). Cells were stained with Annexin-V-PE (Immunostep, Salamanca, Spain) to measure apoptosis commitment. Cells were co-stained together with DCF-DAC and Annexin-V-PE to measure the co-localization of apoptosis and ROS production. Fluorescence was measured by FACScalibur Flow Cytometer (BD Biosciences Clontech) and data were analyzed by CellQuest software.