Fitc conjugated active caspase 3 antibody

Cleaved caspase 3 was detected using FITC conjugated active caspase 3 antibody (BD Biosciences, San Jose, CA) according to the manufacturer's instructions. MCF7 cells were seeded at a density of 3x10⁵ cells/T25 flask and treated with MEAD (15 mg/ml and 20 mg/ml) and Bcl-2 inhibitor ABT-737 (10 μM) for 24 h. The cells were trypsinized, washed twice with cold (1X) PBS and centrifuged at 300 g x 5 min. The cell pellet was resuspended in 0.5 ml BD Cytofix/Cytoperm solution and incubated for 20 min on ice. The cells were then washed twice with BD Perm/Wash buffer (1X) and incubated with 20 μl of FITC conjugated rabbit anti-caspase 3 antibody. Finally the cells were washed with wash buffer and resuspended in a 0.5 ml buffer and analyzed using FACS Aria III flow cytometer (BD Biosciences, San Jose, CA).

FITC conjugated active caspase-3 antibody (BD biosciences, USA) was used to detect the active form of caspase-3 in the cells undergoing apoptosis. Cells were plated in a 6-well culture plate at a density of 0.5 × 10⁶ cells and harvested after 24 and 48 h of incubation with the test compound. The collected cells were washed twice with cold 1X PBS and then resuspended in 0.5 ml of BD Cytofix/Cytoperm solution followed by a 20-min incubation on ice. After incubation, the cells were washed twice in a BD Perm/Wash buffer (1X) and were labelled with 5 µL of FITC rabbit anti-caspase-3 antibodies. The labelled cells were washed again with wash buffer and resuspended in 0.5 ml of buffer and analyzed by acquiring a minimum of 5,000 events on the FACSAria III cell analyzer and sorter.

Detection of cleaved caspase 3 was done using fluorescin isothiocyante (FITC) conjugated active caspase 3 antibody according to the manufacturer's instructions (BD Biosciences, San Jose, CA). Briefly, both OVCAR3 and SKOV3 cells were plated at a seeding density of 1 × 10⁵ cells/T25 cm² tissue culture flask and allowed to attach overnight. Both OVCAR3 and SKOV3 cells were then treated with hWJSC-CM (50%), hWJSC-CL (15 μg/ml), paclitaxel (5 nM) or doxorubicin (30 nM) for 24 h under standard culture conditions. Following the treatment period the cells were trypsinized, centrifuged (1000 rpm × 5 min) and the pellet washed with ice-cold PBS. The cells were again centrifuged and the pellet was resuspended in 0.5 ml of Cytofix/Cytoperm solution and incubated on ice for 20 min. The cells were then washed twice with Perm/Wash buffer (1x) and incubated with 5 μl of FITC conjugated rabbit anti-caspase 3 antibody for 30 min at room temperature. The cells were washed again and resuspended in 300 μl buffer and analyzed using FACS Aria III flow cytometer (BD Biosciences, San Jose, CA) and results computed using FACSDiva^TM software (BD Biosciences, San Jose, CA).