Annexin 5 fitc detection kit

Early and late apoptosis were detected by Annexin-V/PI assay; cells were stained with eBioscience Annexin V-FITC Detection Kit and evaluated for apoptosis by flow cytometry, according to the manufacturer's protocol (Bender System, Wien, Austria). C6 murine cells were washed with PBS and stained with 5 μL of annexin V-FITC in 195 μL of Binding buffer for 10 min at room temperature in the dark. Cells were then washed with PBS and resuspended in 190 μL Binding buffer and stained with 10 μL of PI before reading the sample.
Apoptotic cells were determined using the FACSCalibur equipment (Becton Dickinson), then analyzed with FlowJo, LLC. Both early apoptotic (annexin V-positive, PI-negative) and late apoptotic (annexin V-positive and PI-positive) cells were included in cell death determinations.
Experiments were performed in triplicate and result expressed as mean ± SD. Data were reported as Apoptosis index, calculated as 100 x [(treated – control)/control.

Cells were plated in six-well culture plates for overnight incubation. The half-maximal drug inhibitory concentration (IC50) of DDP in ES-2 cells was measured using the cell counting kit-8 as we previously described, and the IC50 was 52 µM.14 (link) In this study, the experimental and negative control (NC) groups ES-2 cells were treated with 50 µM DDP (Sigma-Aldrich; Millipore) for 48h. Then cells were harvested by trypsinization and washed twice in ice-cold PBS. Apoptosis was assessed via flow cytometry after cells were stained with Annexin V-FITC detection kit (eBioscience, USA). Collected data were analyzed by Flow Jo software (Version 13.0, BD). Each sample was performed in triplicate.