Protocol: Annexin V and PI Staining for Apoptosis by Flow Cytometry
This protocol is intended as a guide only.
A basic apoptosis assay protocol and the required reagents for Annexin V and Propidium iodide (PI) staining, unless otherwise noted, are found in the Annexin V Apoptosis Kit - FITC (Catalog # NBP2-29373)
Materials
- PBS (not included in the kit)
- 1X Binding buffer: 10 mM Hepes pH 7.4, 140 mM NaCl and 2.5 mM CaCl2 (10X dilution before use)
- Positive control cells
- Annexin V-FITC staining solution
- Propidium iodide (PI) staining solution
Methods
- Induce apoptosis by desired method and include vehicle-treated cells/animal (negative control).
- Collect 1-5 x105 cells by centrifugation.
- Wash cells 1X with cold 1X PBS and carefully remove the supernatant.
- Re-suspend the cells in 1X Binding buffer at a concentration of ~1 × 106 cells/mL, preparing a sufficient volume to have 100 µL per sample.
Add 5 μL of Staining solution to tubes as indicated in below table and gently swirl to mix.
Tube # Cells Stain 1 Stabilized Control Cells --- 2 Stabilized Control Cells 5 µL Annexin V-FITC 3 Stabilized Control Cells 5 µL PI Solution 4 Un-induced Experimental Control 5 µL Annexin V-FITC + 5 µL PI 5 Apoptosis Induced Experimental Sample 5 µL Annexin V-FITC + 5 µL PI - Incubate the mixture for 20 minutes at room temperature in the dark.
- Add 400 μl 1X Binding buffer to each tube, gently mix or flick the tube.
- Analyze the cells immediately (within 1 hour) by flow cytometry.
Note: Use the positive control cells (positive for both Annexin V-FITC and PI) to set up compensation and quadrants. Apoptotic cells have a minimal uptake of PI and will appear dimly stained.
- Annexin V negative - PI negative populations are healthy cells.
- Annexin V positive - PI negative populations represent cells in early apoptosis.
- Annexin V positive - PI positive staining indicate cells are in necrosis (post-apoptotic necrosis or late apoptosis).