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P r o t o c o l s

Annexin V Staining Protocol

 

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 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.

 

 

Staining

 

 

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 as described below:

*The optimal amount of PI may range between 210l/test depending on cell type and experimental system. 2 l/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 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).

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 1X Binding Buffer at a concentration of ~1x 10 6 cells/ml.

 

2.

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

 

3.

Add 5-15 g 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 x 10 5 /100 l and still add 5-15 g of recombinant Annexin V to obtain optimal results.

 

4.

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

 

5.

Add 5 l 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 1X Binding Buffer. Dilute 0.5g 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 and incubate at RT for 15 min in the dark.

 

6.

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

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Annexin V Staining Protocol
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APO-DIRECT™ Procedure
Detection of BrdU Incorporation in DNA Synthesizing Cells
Staining Procedure for Flow Cytometric Detection of Human Cyclins
Indirect Immunofluorescence Staining of Human Platelets
Immunofluorescence Staining of Human Cells by Lysed Whole Blood Method
Immunofluorescent Staining of Mouse and Rat Leukocytes
The Uses of Fc Block in Immunophenotyping of Mouse or Rat Leukocytes
ELISA/ELISPOT
Cytokine ELISA
Mouse IgE ELISA Protocol
Immunohistochemistry
Preparation and Staining of Frozen Tissue Sections
Preparation and Staining of Paraffin Sections
Microwave citrate Pretreatment of Paraffin Sections
Immunohistochemistry / Tissue Section Staining
Cell Staining for Immunofluorescence Microscopy
Western Blotting
Western Blotting with Alkaline Phosphatase Conjugates
Western Blotting with Biotinylated Antibodies
Western Blotting with Horseradish Peroxidase Conjugates
Western Blotting with Monoclonal Antibodies
Western Blotting with Rabbit Polyclonal Antibodies
Preparation of Brain Membrane Fractions for Western Blot Analysis
Immunoprecipitation
Immunopurification of Tyrosine Phosphorylated Proteins
Immunoprecipitation with Antibody:Agarose Conjugates
Immunoprecipitation with anti-Phosphotyrosine: Biotin Conjugates
Immunoprecipitation With Soluble Antibodies
Immunoprecipitation of 35S Labeled Cells
RNAse Protection Assay (RPA)
RNase Protection Assay
Total RNA Isolation
Protein Kinase Assay (PKA)
Protein Kinase Assay with an Immunocomplex
Protein Kinase Assay after Denaturation and Renaturation




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