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)

Biotin

1. Unstained cells

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

3. Cells stained with Annexin V-Biotin and SAv-FITC (no PI)

4.Cells stained with PI and SAv-FITC (no Annexin V-Biotin)

Other Staining Controls:

A cell line that can be easily induced to undergo apoptosis should be used to obtain positive control staining. It is important to note that the basal level of apoptosis and necrosis varies considerably within a population. Thus, even in the absence of induced apoptosis, most cell populations will contain a minor percentage of cells that are positive for apoptosis (Annexin V positive, vital dye negative). The untreated population is used to define the basal level of apoptotic and dead cells. The percentage of cells that have been induced to undergo apoptosis is then determined by subtracting the percentage of apoptotic cells in the untreated from the treated population.

Annexin V Blocking

An additional control which may be performed includes preincubation of cell samples with recombinant Annexin V (unconjugated). This serves to block Annexin V-FITC binding sites and thus demonstrates the specificity of Annexin V-FITC staining. The procedure follows.

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 5-15 µg of purified recombinant Annexin V. The amount of purified recombinant Annexin V required to saturate binding sites may vary according to cell type, and stage of apoptosis. In some cases, investigators may also need to reduce the number of cells to 0.5 × 10 5 /100 µl and still add 5-15 µg of recombinant Annexin V to obtain optimal results.

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

5.Add 5 µl of Annexin V-FITC, Annexin V-PE or *Annexin V-Biotin. Gently mix and incubate at RT for 15 min in the dark.

*For Annexin V-Biotin samples only: After 15 min incubation, wash once with 1 ml of 1× Binding Buffer. Dilute 0.5µg 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 and incubate at RT for 15 min in the dark.

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