Protocol: Annexin V and PI Staining for Apoptosis by Flow Cytometry
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
1. Induce apoptosis by desired method and include vehicle-treated cells/animal (negative control).
2. Collect 1-5 x105 cells by centrifugation.
3. Wash cells 1X with cold 1X PBS and carefully remove the supernatant.
4. 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.
5. 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 |
6. Incubate the mixture for 20 minutes at room temperature in the dark.
7. Add 400 μl 1X Binding buffer to each tube, gently mix or flick the tube.
8. 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).