Annexin 5 pi apoptosis kit

Phosphatidylserine externalization and loss of plasma membrane integrity were detected by flow cytometry (FC500MCL, Beckman Coulter, Brea, USA), using the Annexin V/PI apoptosis kit (Lianke, Hangzhou, China). Before that, cells were harvested and washed in sorbitol buffer (1.2 M sorbitol, 0.5 mM MgCl₂, 35 mM K₂HPO₄, pH 6.8), digested with 20 U/ml lyticase (Sigma-Aldrich) at 30 °C for 50 min. Subsequently, cells were washed and resuspended in 500 μl of binding buffer (1.2 M sorbitol, 10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4), then incubated with Annexin V and PI for 10 min at room temperature in the dark.
To quantify intracellular ROS, cells were firstly harvested and resuspended in 500 μl PBS and stained with 5 μg/ml dihydroethidium (DHE) (Cayman Chemical, Ann Arbor, USA) for 30 min in the dark at room temperature [77 (link)]. Cells with red fluorescence [FL-3 channel (488/620 nm)] were detected using the Cytomics FC500MCL flow cytometer (Beckman Coulter).

For analysis of apoptosis, 5×10⁵ NRK-52E cells were cultured on a 6-well plate containing 2 ml serum-free medium. After 24 h, all cells were processed in accordance with the above grouping. NRK-52E cells were collected 24 h post-treatment and stained with Analysis of apoptosis by flow cytometry (FITC)-conjugated annexin V and propidium iodide (PI) according to the manufacturer’s instructions of the apoptosis detection kit (Annexin V/PI Apoptosis kit; Liankebio, Hangzhou, China). Flow cytometry was performed for analysis of apoptosis (FACSAria; BD Biosciences, Heidelberg, Germany).