Annexin 5 cy3 apoptosis detection kit plus

CdCl₂ was obtained from Sigma-Aldrich (Poole, Dorset, UK). Antibodies against Tom40, GAPDH, cytochrome c, Bax, BCL-xl, HO-1 and caspase 3 were obtained from Santa Cruz Biotechnology (Middlesex, UK). Horseradish peroxidase-conjugated goat anti-rabbit antibody was obtained from Bio-Rad laboratory (Hempstead, UK). Polyacrylamide (30 %) was purchased from Seven Biotech Ltd (Worcestershire, UK). Nitrocellulose membranes were purchased from Amersham Biosciences (Amersham, Bucks, UK). Caspase 3 and Calpain activity detection kits were obtained from Calbiochem (Nothingham, UK) and Promega (Southampton, UK), respectively. Annexin V-Cy3 Apoptosis detection Kit Plus (Cat # K202-25) was obtained from BioVision (Mountain View, CA, USA). Protoporphrin IX cobalt chloride (CoPPIX), SnPPIX, Z-DEVD-FMK, and all other chemicals were of the highest grade available and were obtained from Sigma-Aldrich (Poole, Dorset, UK).

The studies of apoptotic and necrotic cell deaths were carried out using Annexin V-Cy3 Apoptosis Detection Kit Plus from BioVision (Mountain View, CA, USA). Briefly, HepG2 cells were treated with 5 and 10 μM CdCl₂ for 24 h. In the induction and inhibition studies, the cells were pre-treated with either 50 μM Z-DEV-FMK for 1 h or 10 μM CoPPIX for 24 h prior to CdCl₂ exposure. After the incubations, the cells were detached using a low trypsin concentration (0.1 %) for one minute. The media containing the cells were centrifuged for 5 min at 1000 x g. The cell pellets were then resuspended in 500 μl binding buffer and 5 μl of annexing- V-Cy3 dye and 1 μl of SYTOX Green dye was added to the resuspended cells and mixed thoroughly. The populations of live, apoptotic and necrotic cells were measured using flow cytometry (EPICS XL Coulter; Beck- man, Indianapolis, IN, USA) according to the manufacturer protocol.