Fitc annexin 5 apoptosis detection kit 1

CD8⁺ T cells were isolated (Miltenyi Biotec) and washed twice with PBS, and centrifuged at 1500 r.p.m. for 5 min to remove supernatant. The apoptotic cell population was examined using a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, CA, USA). T cells were suspended in 200 µl binding buffer and stained with 5 µl Annexin V and PI at room temperature for 15 min in the dark. The T-cell population was analysed by CytoFLEXS flow cytometry according to the instructions governing the use of the BD Apoptosis Detection Kit. CD8⁺ T cells were isolated (Miltenyi Biotec) and washed twice with PBS, and centrifuged at 1500 r.p.m. for 5 min to remove supernatant. The apoptotic cell population was examined using a FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen, CA). T cells were suspended in the 200 µl binding buffer and stained with 5 µl Annexin V and PI at room temperature for 15 min in the dark. The T-cell population was analysed by CytoFLEXS flow cytometry according to the instructions governing the use of the BD Apoptosis Detection Kit.

The extent of apoptosis was determined by flow cytometry using FITC-labelled annexin V and propidium iodide. Following treatment, the A375 cells were harvested and stained according to the instructions of the annexin V-FITC apoptosis detection kit 1 (BD Biosciences, San Diego, CA, USA). The cell samples were then analyzed on the FACS Aria™ Flow Cytometer (BD Biosciences, San Diego, CA, USA). PI signal (emitted at 617 nm) was read on the PE-A channel while the annexin V-FITC signal (emitted at 530 nm) was read on the FITC-A channel. The data was obtained and analyzed using BD FACSDiva™ 6.0 software.

Apoptosis were performed with Annexin V-FITC apoptosis detection kit 1 according to the manufacturer's instructions (BD Biosciences, San Jose, CA). All analyses were performed in triplicate.

Cell viability was determined using the MTT assay. Briefly, after transient transfection, K562 cells were seeded into a 96-well plate at a concentration of 1.5 × 10⁴ cells/100 μL. A total of 24 h after transfection, cells were incubated with 1S,3S RSL3 or 1R,3S RSL3 for an additional 24 h period, and then 10 μL of MTT labeling reagent (Cell Proliferation Kit 1, Roche, Mannheim, Germany) was added to each well, according to the procedures recommended by the manufacturer. The amount of the soluble formazan product in each well was measured by photometric reading at 570/690 nm using a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA). The experiments were repeated in triplicate for each transfection. Cell death analysis was assessed by double-staining with annexin-V and propidium iodide (PI) using an Annexin V-FITC Apoptosis Detection Kit 1 (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol, as previously reported [11 (link)]. A total of 48 h after transfection, cells were analyzed using an Accuri C6 flow cytometer (BD Biosciences) and BD ACCURI C-Flow software.

Cell apoptosis was measured at 48 hr using the Annexin V-FITC Apoptosis Detection Kit-1 (BD Biosciences Pharmingen, San Diego, CA, USA) according to the manufacturer’s protocol. Each sample was assayed in triplicate.

Cell apoptosis was measured at 48 h after treatment using the Annexin VFITC apoptosis Detection Kit-1 (BD Pharmingen, San Diego, CA) according to the manufacturer’s protocol. Each sample was assayed in triplicate. A minimum of 5,000 cells were then analyzed by FACScan with Cell Quest software (Backton Dickinson) for acquisition and analysis.