Tacs annexin 5 fitc

The tetrazolium ring of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) can be reduced with active dehydrogenases to produce a formazan precipitate. Cells were incubated in serum-free media with MTT (0.2 mg/mL) for 45 min at 37 °C. Thereafter, the media were removed and DMSO added to each well to dissolve the formazan precipitate for 20 min with gentle agitation. A total of 100 µL of the supernatants was analyzed in 96-well multiwell plates at 550 nm using a VERSAmax microplate reader (Molecular Devices, San Jose, CA, USA). For Annexin-V-FITC staining, the cells were washed twice with Annexin-V binding buffer (containing 10 mM HEPES-NaOH, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 1.8 mM CaCl₂), resuspended in 100 μl of a 1:100 dilution of TACS Annexin-V-FITC (R&D Systems, Biotechne, Minneapolis, MN, USA; ID 4830-01-K) in Annexin-V binding buffer containing 1 μg/mL Propidium Iodide (PI) and incubated for 15 min at room temperature. Fluorescence was measured with a CytoFLEX flow cytometer (Beckman Coulter, Pasadena, CA, USA), by analyzing at least 10,000 events per condition. The data analysis was performed using Kaluza softwar (Beckman Coulter, Pasadena, CA, USA).