Elabscience® R&D Annexin V Fluorescent Double-Stained Cell Apoptosis Detection kit was used to identify apoptotic and necrotic cells.
Annexin V is a member of the annexin family of intracellular proteins that bind to phosphatidylserine (PS) in a calcium-dependent manner. Annexin V-Flouorescent, which bind to the membrane of apoptotic cells through PS exposed outside cells, can detect apoptosis by flow cytometry or fluorescence microscopy
Specifical Staining Buffer which can specifically bind to double-stranded DNA and produces strong fluorescence, and it's normally fails to penetrate cell membranes. Due to late apoptotic or necrotic cells loss of integrity of membrane, Specifical Staining Buffer can enter the cells to stain DNA. Cells at different apoptotic stages can be distinguished by using with Annexin V and Specifical Staining Buffer.
1. Suspension Cells
A. Suspension cells were induced to apoptosis according to the experimental scheme. Centrifuge at 300 g for 5 min, discard the supernatant. Add appropriate Cell Staining Buffer [Cat: E-CK-A171, please contact the local distributors] to re-suspend gently and count the cells.
B. Take 1-5 × 105 re-suspend cells in step1. Centrifuge at 300 g for 5 min, discard the supernatant. Add appropriate Cell Staining Buffer [Cat: E-CK-A171] to wash the cells, centrifuge at 300 g for 5 min, discard the supernatant. Add 100 μL of 1 × Annexin V Binding Buffer to re-suspend the cells.
C. Add 5 μL of Annexin V-Fluorescent Staining Buffer and 5 µl of DNA dye (PI or 7-AAD) to each tube.
D. Gently vortex the cells and incubate at room temperature for 15-20 min in the dark.
E. Analyze the cells immediately with proper machine settings. Otherwise, place the cells on ice in the dark and analyze within 1 h.