Annexin 5 fitc pi kit

HeLa cervical cancer cells were purchased from Highveld Biological, Johannesburg, South Africa. RPMI 1640 cell culture medium and fetal bovine serum was purchased from GE Healthcare Life Sciences (Logan, UT, USA). Trypsin-EDTA, Dulbecco’s phosphate buffered saline (DPBS) with Ca²⁺ and Mg²⁺ and Dulbecco’s phosphate buffered saline (DPBS) without Ca²⁺ and Mg²⁺ were purchased from Lonza (Wakersville, MA, USA). Trypan blue, bisBenzamide H 33,342 trihydrochloride (Hoechst 33342), cisplatin, penicillin/streptomycin and bovine serum albumin fraction V were purchased from Sigma-Aldrich (St. Louis, MI, USA). NucRed^TM Live 647, CellRox^® Orange reagent, Lysotracker™ Deep Red and Tetramethylrhodamine ethyl ester (TMRE) were purchased from Molecular Probes^®-Life Technologies-Thermo Fisher Scientific (Logan, UT, USA). Annexin V-FITC/PI kit was purchased from MACS Miltenyi Biotec (Cologne, Germany). Cleaved caspase 3 (Asp175) (D3E9) Rabbit mAb, Cleaved caspase 8 (Asp391) (18C8) Rabbit mAb, Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa fluor^® 647 conjugate), Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa fluor^® 488 conjugate) and Phosphorylated Histone H3 were purchased from Cell Signaling Technology (Danvers, MA, USA).

Phosphatidylserine translocation (PS) was detected after 24 and 48 h using the Annexin V-FITC/PI kit from MACS Miltenyi Biotec with modifications. Treatment medium was removed, and cells were stained using a mixture of Annexin V-FITC (final concentration of 1× the stock) and Hoechst 33342 (final concentration of 2 μg/mL) for 15 min at room temperature. PI (final concentration of 1 μg/mL) was added to the Annexin/Hoechst 33342 stain in the 96-well plate just before image acquisition.

On day 8 of GC culture, cells were thoroughly washed twice with phosphate buffered saline (PBS) and then trypsinized using 250 μl TryplE solution (Thermo Fischer, USA) at 37°C for 20 min. Post trypsinization, all detached cells were centrifuged and resuspended in 1 ml MEM and analyzed for viability and apoptosis using an Annexin-V FITC/PI kit (Miltenyi biotec, Germany). Cells were centrifuged and pellets were re-suspended in 100 μl of binding buffer to which 10 μl of Annexin V reagent was added and kept for incubation in the dark for 15 min. followed by washing and resuspension in 500 μl binding buffer. Next, 5 μl of PI (Propidium iodide, 500 μg/ml) was added to the cells with gently mixing just prior to flow cytometric analysis. The fluorescence signal was quantified from single cells (10,000 counts) by a flow cytometer (Gallios, Beckman-Coulter, Germany) and the data obtained was analyzed using the Kaluza-software (Beckman-Coulter, Germany).