Apc annexin 5 and pi

Cells undergoing apoptosis or necrosis can be stained and quantified using Annexin V and propidium iodide (PI). Cells treated with H₂O₂, were washed once with phosphate buffered saline (PBS), and stained for 15 min with allophycocyanin-conjugated (APC)–Annexin V and PI (Becton Dickinson), according to the manufacturer’s instructions. Quantification of apoptotic/necrotic cell-death staining with APC–annexin V and PI under each condition were measured with a BD LSRFortessa™ apparatus (Becton Dickinson) and analyzed using FlowJo ™ Software (Tree Star Inc. Ashland, OR, USA). Annexin V-negative/PI-negative cells were considered living cells.

For cell cycle analysis, the cells were harvested and fixed with ice-cold ethanol overnight at 4°C. After washing twice with PBS, the cells (10⁶ cells/tube) were treated with RNase A and stained with propidium iodide (PI) (Sigma) in the dark, and then analyzed by flow cytometry (FACScan; BD). In addition, the cultured cells (10⁵ cells /tube) were harvested and stained in duplicate with APC annexin V and PI (BD) to characterize spontaneous cell apoptosis according to the manufacturer’s instructions. Each experiment was performed in triplicate.

For apoptosis analysis, quantification of apoptotic cells was performed with double fluorescence staining with APC Annexin V and PI (BD Biosciences, San Jose, CA, USA) with subsequent flow cytometry analysis according to the manufacturer’s instructions. The relative proportion of Annexin V-positive and PI-negative cells was determined using the ModFitLT software (Becton Dickinson, San Diego, CA, USA) and counted as early apoptotic cells (Annexin V-positive, PI-negative), late apoptotic cells (Annexin V-positive, PI-positive), necrotic cells (Annexin V-negative, PI-positive), and viables (Annexin V-negative, PI-negative). The experiments were conducted three times independently.