Fitc conjugated annexin 5 pi assay kit

The NCI-H929 cells (1 × 10⁶ cells/well) were treated with secalonic acid for 24 h and 48 h (in humidified 5% CO₂ atmosphere at 37 °C), and apoptosis/necrosis was assessed by the flouresceinisothiocynate (FITC)-conjugated annexin V/PI assay kit (eBioscience™Annexin V; Invitrogen, Germany) by flow cytometry. Briefly, treated cells were centrifuged at 1200 rpm for 5 min and washed twice with ice-cold PBS (phosphate-buffered saline) and 1 × binding buffer, respectively. After the cells were re-suspended in binding buffer at a total number of 4 × 10⁶ cells/mL, 5 µL of FITC-conjugated annexin V was added to 100 µL of the cell suspension and incubated for 15 min at room temperature. Then, the cells were washed with 1 × binding buffer and resuspended in 200 µL of 1 × binding buffer. Propidium iodide staining solution (5 µL) was added and analyzed after 15 min incubation at room temperature in the dark using BD Accury C6 Flow Cytometer (BD Biosciences). Apoptosis and necrosis were evaluated on fluorescence 2 (FL3 for propidium iodide) versus fluorescence 1 (FL1 for annexin) plots. The fluorescent cells % in each quadrant pointed out different cell populations corresponding to viable and non-apoptotic (annexin V−PI−), early apoptotic (annexin V+PI−), late apoptotic as well as early necrotic (annexin V+PI+) and late necrotic (annexin V−PI+) cells.

To detect the apoptotic CCRF-CEM cells resulting from the application of BTL, compound 8, doxorubicin, or DMSO, the annexin V/propidium iodide (PI) staining combined with the flow cytometry was used in similar experimental conditions as reported earlier [33 (link), 41 –43 (link)]. The concentration of samples used and CCRF-CEM cells were those as reported above under the standard cell culture conditions. To detect the apoptotic cells, the fluorescein isothiocyanate (FITC)-conjugated annexin V/PI assay kit (eBioscience™ Annexin V; Invitrogen, San Diego, USA) was applied to treated cells after 24 h incubation followed by the measurements with BD Accury C6 Flow Cytometer (BD Biosciences) [42 (link), 43 (link)]. The assays were performed separately three times and in triplicate.

Apoptosis was measured by using the FITC-conjugated Annexin V/PI assay kit (Invitrogen, Carlsbad, CA, USA) and flow cytometry. After treatment with scramble RNA or siRNA STAT1 for 24 h, cells were treated with 10 μM MG132 for 24 h and then collected according to the manufacturer’s protocol.