Before you start
- Collect floating cells as well as adherent cells.
- Prepare single-stained controls and an apoptosis-positive control.
Reagents and materials
Totals are for 8 samples (+10% extra). Per-sample amounts are in the middle column.
| Reagent | Per sample | Total |
|---|---|---|
| Cold PBS (wash) | 1 mL | 1 mL |
| 1× Annexin V binding buffer | 0.5 mL | 0.5 mL |
| Fluorescent Annexin V | 5 µL | 5 µL |
| Propidium iodide or 7-AAD (per kit) | 5 µL | 5 µL |
Buffers and recipes
1× Annexin V binding buffer per 100 mL
| HEPES (10 mM) | 0.24 g |
| NaCl (140 mM) | 0.82 g |
| CaCl₂, anhydrous (2.5 mM) | 0.028 g |
| Ultrapure water | to 100 mL |
Adjust to pH 7.4. Calcium is required for Annexin V binding.
Equipment
- Flow cytometer
- FACS tubes
Procedure
Tick steps as you go (saved in this browser). Steps with a clock have a built-in timer.
Harvest
Collect cells (including supernatant). Detach adherent cells gently; avoid EDTA or wash it out thoroughly.
Wash
Wash twice in cold PBS.
Resuspend
Resuspend 1 × 10⁵ – 1 × 10⁶ cells in 100 µL binding buffer.
Stain
Add Annexin V and PI, mix and incubate 15 min at room temperature in the dark.
Dilute and acquire
Add 400 µL binding buffer and acquire within 1 h without washing.
Gate
Annexin V⁻PI⁻ live; Annexin V⁺PI⁻ early apoptotic; Annexin V⁺PI⁺ late apoptotic or necrotic.
Critical points
- No EDTA in buffers — it chelates the calcium Annexin V needs.
- PI-containing samples cannot be fixed.
Troubleshooting
| Problem | What to check or change |
|---|---|
| Many Annexin⁺ in controls | Gentler harvesting; trypsin and scraping expose phosphatidylserine. |
| No staining | Check calcium in buffer and reagent expiry. |
Safety
PI is a DNA intercalator and possible mutagen: wear gloves.
Further reading
- Vermes I et al. A novel assay for apoptosis: flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods (1995).
This is a general starting-point protocol written for SolvesEverything. Follow your institution’s approved protocols, safety rules and manufacturers’ instructions, and optimise for your system. Spotted an error? Tell us.