etry; Ca2+ ionophore and a chelating agent EGTA served as the positive and negative control respectively. undergoing late apoptosis or necrotic cells. Briefly, U937 cells were incubated with a near IC50 concentration of MAL-A at 37uC, 5% CO2 for 0 24 h. After two washes, cells were resuspended in Annexin V binding buffer and Annexin VFITC was added according to the manufacturers’ instructions. The cells were incubated for 30 minutes in the dark at 37uC and just prior to acquisition, PI was added and cells were acquired in a flow cytometer. Determination of caspase activity Activity of caspases-8, 9 and 3 was detected in cell lysates as per the manufacturer’s instructions. Briefly, U937 cells after incubation with MAL-A was washed twice with ice cold PBS, cell lysates prepared and protein concentration estimated. Lysates were combined with 50 ml of 26 reaction buffer, caspase 3 substrate DEVD-pNA or caspase 8 substrate IETD-pNA or caspase 9 substrate LEHD-pNA following incubation at 37uC for 03 h; the release of chromophore para nitroanilide was quantified by measuring absorbances at 405 nm every 30 minutes for 3 h. To establish whether MAL-A induced death was caspase independent or not, U937 cells were preincubated with a pan caspase inhibitor Z-VAD-FMK followed by 48 h co-incubation with MAL-A and cell viability evaluated by the MTS-PMS assay as described above. Analysis of mitochondrial transmembrane potential The mitochondrial transmembrane electrochemical VS-4718 price gradient was measured using JC-1,. JC-1, a cell permeable, cationic, lipophilic dye freely crosses the mitochondrial membrane and forms J-aggregates which fluoresce red; accordingly, viable cells with a normal mitochondrial membrane potential when stained with JC-1 exhibit a pronounced red fluorescence. Following an apoptotic stimulus, the resultant decrease in the mitochondrial membrane potential prevents JC-1 from entering the mitochondria and remains as monomers in the cytosol that emits a predominantly green fluorescence. Therefore, the 
ratio of J-aggregates/monomers serves as an effective indicator of the cellular mitochondrial transmembrane potential, allowing apoptotic cells to be easily distinguished from their non-apoptotic counterparts. Briefly, U937 cells following incubation with MAL-A, were stained with JC-1. Cells were then acquired in a flow cytometer on the basis of quadrant plot to distinguish monomers from J-aggregates and analyzed using Cell Quest Pro software. To set the quadrants, cells were treated with H2O2, representative of cells with depolarized mitochondrial membrane potential. Immunoblotting Cells were resuspended in cell lysis buffer and after sonication, centrifuged and protein concentration measured. Samples were boiled with sample buffer for 5 minutes and stored at 220uC. Lysates were resolved by 10% SDS-PAGE and transferred onto nitrocellulose membranes. After blocking the non specific binding sites with 5% non fat dried milk in Tris buffered saline for 2 h, membranes were incubated overnight at 4uC with antibodies against poly polymerase and cytochrome c in TBS containing 5% BSA. Membranes were washed thrice with TBS containing 0.1% Tween 20 and binding detected with HRP conjugated secondary antibody, the bands being visualized using diaminobenzidine and analyzed using G-BOX gel doc apparatus. Measurement of cardiolipin oxidation To determine the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/22187349 effect of MAL-A induced ROS upon cardiolipin peroxidation in mitochondria, 10-N-nonyl acridine orange w
