Fitc labeled recombinant annexin 5 apoptosis detection kit

Flow cytometry was performed using a FITC-labeled recombinant Annexin V apoptosis detection kit (Beyotime, China). Either treated or untreated cells were harvested continuously, washed in PBS, and reconstituted in a coupling buffer (10 mM HEPES/NaOH, pH 7.4, 2.5 mM CaCl₂, 140 mM NaCl). Annexin V-FITC was introduced (at 250 ng/mL final concentration) before incubation in the darkness at 4 °C for 15 min, followed by washing with PBS and resuspension in 190 µL coupling buffer. Then, 10 µL of propidium iodide (PI) was added for 5 min. Stained cells were examined by a FACStar plus flow cytometer (Becton Dickinson, USA). The ratio of fluorescence intensities that were excited at 488 nm was monitored at an emission wavelength of 515 nm and 560 nm for FITC and PI, respectively. The collected data were analyzed with a BD BioSciences FACSCalibur flow cytometer using CellQuest software.

A FITC-labeled recombinant Annexin V Apoptosis Detection kit (Beyotime Institute of Biotechnology) was used to measure apoptosis. Cells were seeded in six-well plates (5.0×10⁴ cells/well) and cultured at 25°C overnight prior to treatment. After treatment of IL-1β, IL-1β + CT, IL-1β + IWR-1- endo and IL-1β + CT + IWR-1-endo, for 48 h, cells were centrifuged at 1,000 × g for 5 min at 4°C. Supernatants were subsequently removed. Cells were incubated with 5 µl Annexin V-FITC at 4°C for 15 min in darkness, followed by 5 µl propidium iodide (PI) for a further 5 min. The rate of early apoptosis was detected using Accuri C6 FACScan flow cytometer with CFlow Plus^® version 1.0 software (BD Bioscience). Early apoptotic cells were counted in the lower right quadrant of the display.