Fitc conjugated annexin 5 and pi annexin 5 fitc apoptosis detection kit

HuTu-80 cells were seeded in 6-well plates at a density of 5 × 10⁵ cells per well and incubated for 24 h under standard conditions. Then the medium was replaced with fresh medium containing compound 9 at 8 µM. In order to determine induction of apoptosis, cells were harvested at 6, 18 and 24 h post treatment. Cells were detached with trypsin, washed with cold PBS and then resuspended gently in binding buffer at concentration of 10⁶ cells/mL. Thereafter, cells were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and PI (Annexin V-FITC Apoptosis Detection Kit, Millipore, Bedford, MA, USA) for 15 min in the dark and subsequently analyzed using NovoCyte Flow Cytometer (ACEA Biosciences Inc., San Diego, CA, USA). For each sample, 10.000 events were acquired.

RLS₄₀ cells were seeded in 24-well plates at a density of 1.5 × 10⁶ cells per well and incubated in antibiotic-free IMDM supplemented with 10% FBS (Sigma-Aldrich, St Louis, MO, USA) in the presence of binase (500 µg/mL) for 48 h under standard conditions. The medium was discarded and the cells were gently suspended in a binding buffer at a concentration of 10⁶ cells/mL. Then, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and PI (Annexin V-FITC Apoptosis Detection Kit, Millipore, Bedford, MA, USA) for 15 min in the dark and immediately analysed using a NovoCyte flow cytometer (ACEA Biosciences Inc., San Diego, CA, USA).