A general protocol for staining cell for cytometry analysis

General Annexin V Staining Procedure

Solutions

1.10X Binding Buffer (Cat. No. 66121A):0.1 M HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl 2 . Dilute to 1× prior to use.

2. Propidium Iodide (PI). Prepare a 50 µg/ml stock solution of PI in 1× 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).

4.1× 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.

 Staining

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

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

3. Add Annexin V and Vital Dye as described below:

*The optimal amount of PI may range between 2–10µl/test depending on cell type and experimental system. 2 µl/test is the recommended starting amount.

4.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 1× Binding Buffer. Dilute 0.5 µg Streptavidin-Fluorescein Isothiocynate (SAv-FITC, Cat. No. 13024D) into 100 µl of 1× Binding Buffer and add to the cell pellet. Gently mix the cells. Add 2 µl PI and incubate for 15 min at RT.

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

Note: Methods for utilizing Annexin V binding on adherent cells ( i.e., monolayer) have been described by van Engeland et al. 16 and Casciola-Rosen et al.1 However, these methods are not performed as a routine quality control for the Annexin V-FITC Apoptosis Detection Kit I and Kit II.

Suggested Controls for Flow Cytometric Analysis of Annexin V samples.

The following controls are used to set up compensation and quadrants:

FITC

1.Unstained cells

2. Cells stained with Annexin V-FITC alone (no PI)

3. Cells stained with PI alone (no Annexin V-FITC)

PE

1. Unstained cells

2.Cells stained with Annexin V-PE alone (no 7-AAD)

3.Cells stained with 7-AAD alone (no Annexin V-PE)