Annexin 5 pe 7 aad detection kit

Flow cytometry was used to measure OGD/R-induced apoptosis of RGCs using a Propidium Iodide (PI)/Annexin V-FITC Detection Kit and an Annexin V-PE/7-AAD Detection Kit (BD Biosciences, USA) according to the manufacturer’s instructions. Flow cytometric analysis using FlowJo 7.6.2 (FlowJo, LLC) was performed following standard protocols. Annexin V⁺ cells were identified as apoptotic ones. Ten thousand cells were collected and analyzed in each flow cytometric assay. Six independent experiments were performed.

Induction of apoptosis was evaluated following Annexin V staining of adherent cells protocol for flow cytometry. Briefly, B16 cells were cultured in a 6-well plate (2.5x10⁵cells/well) in 2 mL standard cell culture medium. After 18h medium was replaced and 100 μg of BLS were added. Forty eight hours later, apoptosis was assessed using the Annexin V-PE/7-AAD detection kit (#559763, BD Pharmingen, San Diego, CA, USA). Cells were gently detached using Accutase Cell Detachment Solution (BD Biosciences; San José, CA, USA) and fluorescence-activated cell sorter analysis was performed, as described below.

The percentage of apoptosis was evaluated using an Annexin V-PE/7-AAD detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer's protocol.
In vitro experiment, cells were cultured in the presence of 0, 100, 200 and 400 μmol/l NaF for 24 and 48 h. Prior to toxicity detection, the cells were collected and washed two times with phosphate buffered saline (PBS, PH 7.4). Pellets were collected and resuspend in 100 μl PBS, and stained with PE Annexin V and 7-amino-actinomycin (7-AAD) for 15 minutes in the dark. Then, 400 μL binding buffer (BD Pharmingen) was added. Data were then obtained by a FACSCalibur (Becton Dickinson, USA).
In vivo experiment, mice were humanely killed at 21 and 42 days of age, spleens were taken from each mouse and ground to form a cell suspension that was filtered through a 300-mesh nylon screen. The cells were washed twice with cold PBS (phosphate buffer solution, pH 7.2-7.4) and then suspended in PBS at a concentration of 1×10⁶ cells/mL. 100 μL portions of the cell suspension were transferred into 5 mL culture tubes, and stained with PE Annexin V and 7-amino-actinomycin (7-AAD). The mixture was gently vortexed and incubated for 15 min in the dark. 400 μL of 1×binding buffer was added to each tube, and analysis by FACSCalibur (BD FACSCalibur).