Annexin 5 fluorescein isothiocyanate fitc propidium iodide kit

Cell apoptosis rate was analyzed using a commercial Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (Beyotime, Shanghai, China). After transfection for 72 h, chordoma cells were collected and incubated with Annexin V-FITC and PI for 30 min in the darkroom at room temperature. Cell fluorescence was measured on the Influx Flow Cytometer & Cell Sorter System (BD Biosciences, Franklin Lakes, New Jersey, USA). The apoptosis rate (the percentage of cells with FITC⁺ and PI^+/−) was analyzed.

Lipopolysaccharide‐induced chondrocytes were seeded into 6‐well plates (Corning) with 1 × 10⁵ cells per well for 24 h and analyzed using an annexin V‐fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (Beyotime) on flow cytometry. In short, after LPS stimulation or no stimulation, the adherent cells were harvested, washed, and stained in FITC‐annexin V and PI for 1 h in the dark. Then, the fluorescence was analyzed on a flow cytometer (Beckman‐Counter Electronics, Jiangsu, China). Apoptotic cells were sorted in annexin V+/PI− and au8 nnexin V+/PI+ quadrants, and the apoptotic rate was calculated using the following equation: apoptotic rate (%) = 100% × apoptotic cells/total cells.

The CellTiter-Glo^® 3D Cell Viability Assay (promega, Beijing, china), a test system to detect the amount of present ATP proportional to the cell number, was employed to estimate the cell proliferation. Later, the CellTiter-Glo^® 3D reagent was added into each well at the equivalent volume of cell culture medium in the well. Then, the contents were mixed vigorously for 5 min to induce cell lysis, and the plate was incubated at room temperature for an additional 25 min to stabilize the luminescent signal. Afterwards, 200 µL supernatants were transferred in technical replicates into the 96-well opaque-walled plate and the luminescence was measured. In cell apoptosis assay, both 3D and 2D MDA-MB-231 cells were cultured in the serum-free medium before trypsin digestion. Then, cells were stained using the annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (Beyotime, Beijing, China) according to the manufacturer’s instructions. Briefly, after trypsin digestion in the absence of EDTA, 5×10⁵ cells were collected and washed twice with the precooled PBS, and then 100 μL binding buffer was added for cell resuspension. Later, 5 μL annexin V-FITC and 10 μL PI standing solutions were mixed gently in dark at room temperature for 5 min, and the apoptosis level was measured by flow cytometry.

Apoptotic rate of transfected HL-60 and KG-1 cells was analyzed by Annexin V-Fluorescein isothiocyanate (FITC)/Propidium Iodide (PI) kit (Beyotime, Shanghai, China) on flow cytometry. After transfection for 48 hours, apoptotic cells were collected and washed with phosphate-buffered saline (PBS). Cell suspension of 10⁶ cells were prepared and labelled with FITC-Annexin V and PI for 30 minutes in the dark. The fluorescence was analyzed on an Influx Flow Cytometer & Cell Sorter System (BD, Franklin Lakes, NJ, USA). Quadrants were positioned on Annexin V/PI plots to distinguish apoptotic cells (Annexin V+/PI-, Annexin V+/PI+). Apoptotic rate=apoptotic cells/total cells×100%.

After 4 Gy irradiation, cells were cultured under normal conditions for 24 h. Then, cell apoptosis was measured using an annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) kit (Beyotime) according to the manufacturer’s instructions. Cells were cultured in 6-well plates and incubated overnight. After the indicated treatment, cells were collected and stained with annexin V-FITC for 5 min and 5 μL PI for 5 min at 4 °C. Samples were analysed by flow cytometry on a BD FACS Aria flow cytometry system (BD Biosciences, Franklin Lakes, USA).