Annexin V Staining Protocol

Friday, October 17, 2003

Description
Annexin V Staining Protocol

Procedure

1.
Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of ~1 x 10 6 cells/ml.

2.
Transfer 100 l of the solution (~1 x 10 5 cells) to a 5 ml culture tube.

3.
Add Annexin V and Vital Dye

Gently mix the cells and incubate for 15 min at RT in the dark.

*For Annexin V-Biotin samples only: After 15 min incubation, wash once with 1 ml of 1X Binding Buffer. Dilute 0.5 g Streptavidin-Fluorescein Isothiocynate (SAv-FITC, Cat. No. 13024D) into 100 l of 1X Binding Buffer and add to the cell pellet. Gently mix the cells. Add 2 l PI and incubate for 15 min at RT.

5.
Add 400 l of 1X Binding Buffer to each tube. Analyze by flow cytometry as soon as possible (within 1 hr).

Recipes
Reagents from BD Pharmingen

10X Binding Buffer (Cat. No. 66121A) : 0.1 M HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl 2 . Dilute to 1X prior to use.

2.
Propidium Iodide (PI). Prepare a 50 g/ml stock solution of PI in 1X PBS buffer. Store at RT. Recommended for use in parallel with Annexin V-FITC (Cat. No. 65874X, 65874H) or Annexin V-Biotin (Cat. Nos. 65872X, 65872H).

3.
Via-Probe (Cat. No. 34321X): A convenient ready-to-use solution of the nucleic acid dye 7-AAD. Recommended for use in parallel with Annexin V-PE (Cat. No. 65875X, 65875H) or Annexin V-Biotin (Cat. Nos. 65872X, 65872H).

4.
1X PBS Buffer: 8 g NaCl; 0.2 g KCl; 1.44 g Na 2 HPO 4 7H 2 0; 0.24 g KH 2 PO 4 ; H 2 0 to 1 liter. Adjust pH to 7.2, autoclave and store at RT.

Supplies

Tips

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Annexin V Staining Protocol