Technical Data Sheet

PE Annexin V Apoptosis Detection Kit I

Product Information

Material Number: 559763

Component: 51-66121E

Description: 10X Annexin V Binding Buffer

Size: 50 ml （1 ea）

Storage Buffer: Aqueous buffered solution containing no preservative.

Component: 51-68981E

Description: 7-AAD

Size: 2.0 ml （1 ea）

Vol. per Test: 5 μl

Storage Buffer: Aqueous buffered solution containing fetal bovine serum ａnd ≤0.09% sodium

azide.

Component: 51-65875X

Description: PE Annexin V

Size: 0.5 ml （1 ea）

Vol. per Test: 5 μl

Storage Buffer: Aqueous buffered solution containing BSA ａnd ≤0.09% sodium azide.

Description

Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenence of tissue homeostasis. The

apoptotic program is characterized by certain morphologic features, including loss of plasma membrane asymmetry ａnd attachment,

condensation of the cytoplasm ａnd nucleus, ａnd internucleosomal cleavage of DNA. Loss of plasma membrane is one of the earliest features.

In apoptotic cells, the membrane phospholipid phosphatidylserine （PS） is translocated from the inner to the outer leaflet of the plasma

membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35-36 kDa Ca2+ dependent phospholipid-binding

protein that has a high affinity for PS, ａnd binds to cells with exposed PS. Annexin V may be conjugated to fluorochromes including

Phycoerythrin （PE）. This format retains its high affinity for PS ａnd thus serves as a sensitive probe for flow cytometric analysis of cells that are

undergoing apoptosis. Since externalization of PS occurs in the earlier stages of apoptosis, PE Annexin V staining can identify apoptosis at an

earlier stage than assays based on nuclear changes such as DNA fragmentation.

PE Annexin V staining precedes the loss of membrane integrity which accompanies the latest stages of cell death resulting from either

apoptotic or necrotic processes. Therefore, staining with PE Annexin V is typically used in conjunction with a vital dye such as

7-Amino-Actinomycin （7-AAD） to allow the investigator to identify early apoptotic cells （7-AAD negative, PE Annexin V positive）. Viable

cells with intact membranes exclude 7-AAD, wheras the membranes of dead ａnd damaged cells are permeable to 7-AAD. For example, cells

that are considered viable are PE Annexin V ａnd 7-AAD negative; cells that are in early apoptosis are PE Annexin V positive ａnd 7-AAD

negative; ａnd cells that are in late apoptosis or already dead are are both PE Annexin V ａnd 7-AAD positive. This assay does not distinguish

between cells that have undergone apoptotic death versus those that have died as a result of a necrotic pathway because in either case, the dead

cells will stain with both PE Annexin V ａnd 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from PE

Annexin V ａnd 7-AAD negative （viable, or no measurable apoptosis）, to PE Annexin V positive ａnd 7-AAD negative （early apoptosis,

membrane integrity is present） ａnd finally to PE Annexin V ａnd 7-AAD positive （end stage apoptosis ａnd death）. The movement of cells

through these three stages suggests apoptosis. In contrast, a single observation indicating that cells are both PE Annexin V ａnd 7-AAD

positive, in of itself, reveals less information about the process by which the cells underwent their demise.

559763 Rev. 8 Page 1 of 3

Flow Cytometric Analysis of PE Annexin V staining. Jurkat cells

（Human T-cell leukemia; ATCC TIB-152） were left untreated （top

panels） or treated for 4 hours with 4 μM Camptothecin （bottom

panels）. Cells were incubated with PE Annexin V in a buffer

containing 7-Amino-Actinomycin （7-AAD） ａnd analyzed by flow

cytometry. Untreated cells were primarily PE Annexin V ａnd 7-AAD

negative, indicating that they were viable ａnd not undergoing

apoptosis. After a 4 hour treatment （bottom panels）, there were

primarily two populations of cells: Cells that were viable ａnd not

undergoing apoptosis （PE Annexin V ａnd 7-AAD negative）; cells

undergoing apoptosis （PE Annexin V positive ａnd 7-AAD negative）.

A minor population of cells were observed to be PE Annexin V and

7-AAD positive, indicating that they were in end stage apoptosis or

already dead.

Preparation ａnd Storage

Store undiluted at 4°C ａnd protected from prolonged exposure to light. Do not freeze.

Application Notes

Application

Flow cytometry Routinely Tested

Recommended Assay Procedure:

PE Annexin V is a sensitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces （Kd of ~5 x 10^-2） with a

higher affinity for phosphatidylserine （PS） than most other phospholipids. PE Annexin V binding is calcium dependent ａnd defined calcium and

salt concentrations are required for optimal staining as described in the PE Annexin V Staining Protocol. Investigators should note that PE

Annexin V flow cytometric analysis on adherent cell types （e.g HeLa, NIH 3T3, etc.） is not routinely tested as specific membrane damage

may occur during cell detachment or harvesting. Methods for utilizing Annexin V for flow cytometry on adherent cell types, however,

have been previously reported （Casiola-Rosen et al. ａnd van Engelend et al.）.

INDUCTION OF APOPTOSIS BY CAMPTOTHECIN

The following protocol is provided as an illustration on how PE Annexin V may be used on a cell line （Jurkat）.

Materials

1. Prepare Camptothecin stock solution （Sigma-Aldrich Cat. No. C-9911）: 1 mM in DMSO.

2. Jurkat T cells （ATCC TIB-152）.

Procedure

1. Add Camptothecin （final conc. 4-6 μM） to 1 x 10^6 Jurkat cells.

2. Incubate the cells for 4-6 hr at 37°C.

3. Proceed with the PE Annexin V Staining Protocol to measure apoptosis.

PE ANNEXIN V STAINING PROTOCOL

PE Annexin V is used to quantitatively determine the percentage of cells within a population that are actively undergoing apoptosis. It relies on

the property of cells to lose membrane asymmetry in the early phases of apoptosis. In apoptotic cells, the membrane phospholipid

phosphatidylserine （PS） is translocated from the inner leaflet of the plasma membrane to the outer leaflet, thereby exposing PS to the external

environment. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for PS, ａnd is useful for identifying

apoptotic cells with exposed PS. 7-Amino-Actinomycin （7-AAD） is a standard flow cytometric viability probe ａnd is used to distinguish viable

from nonviable cells. Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead ａnd damaged cells are permeable to

7-AAD. Cells that stain positive for PE Annexin V ａnd negative for 7-AAD are undergoing apoptosis. Cells that stain positive for both PE

Annexin V ａnd 7-AAD are either in the end stage of apoptosis, are undergoing necrosis, or are already dead. Cells that stain negative for both PE

Annexin V ａnd 7-AAD are alive ａnd not undergoing measurable apoptosis.

559763 Rev. 8 Page 2 of 3

Reagents

1. PE Annexin V （component no. 51-65875X）: Use 5 μl per test.

2. 7-Amino-Actinomycin （7-AAD） （component no. 51-68981E） is a convenient, ready-to-use nucleic acid dye. Use 5 μl per test.

3. 10X Annexin V Binding Buffer （component no. 51-66121E）: 0.1 M Hepes/NaOH （pH 7.4）, 1.4 M NaCl, 25 mM CaCl2. For a 1X working

solution, dilute 1 part of the 10X Annexin V Binding Buffer to 9 parts of distilled water.

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 μl of the solution （1 x 10^5 cells） to a 5 ml culture tube.

3. Add 5 μl of PE Annexin V ａnd 5 μl 7-AAD.

4. Gently vortex the cells ａnd incubate for 15 min at RT （25°C） in the dark.

5. Add 400 μl of 1X Binding Buffer to each tube. Analyze by flow cytometry within 1 hr.

SUGGESTED CONTROLS FOR SETTING UP FLOW CYTOMETRY

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

1. Unstained cells.

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

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

Other Staining Controls:

A cell line that can be easily induced to undergo apoptosis should be used to obtain positive control staining with PE Annexin V and/or PE

Annexin V ａnd 7-AAD. 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 （PE Annexin V

positive, 7-AAD negative or PE Annexin V positive, 7-AAD positive）.

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 population from percentage of apoptotic cells in the

treated population. Since cell death is the eventual outcome of cells undergoing apoptosis, cells in the late stages of apoptosis will have a damaged

membrane ａnd stain positive for 7-AAD as well as for PE Annexin V. Thus the assay does not distinguish between cells that have already

undergone an apoptotic cell death ａnd those that have died as a result of necrotic pathway, because in either case the dead cells will stain with

both PE Annexin V ａnd 7-AAD.

Product Notices

This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-μl experimental

sample （a test）.

1.

2. Source of all serum proteins is from USDA inspected abattoirs located in the United States.

Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before

discarding to avoid accumulation of potentially explosive deposits in plumbing.

3.

4. Please refer to www.bdbiosciences.com/pharmingen/protocols for technical protocols.

References

Andree HA, Reuingsperger CP, Hauptmann R, Hemker HC, Hermens WT, Willems GM. Binding of vascular anticoagulant alpha （VAC alpha） to planar

phospholipid bilayers. J Biol Chem. 1990; 265（9）:4923-4928. （Biology）

Casciola-Rosen L, Rosen A, Petri M, Schlissel M. Surface blebs on apoptotic cells are sites of enhanced procoagulant activity: implications for coagulation events

and antigenic spread in systemic lupus erythematosus. Proc Natl Acad Sci U S A. 1996; 93（4）:1624-1629. （Biology）

Homburg CH, de Haas M, von dem Borne AE, Verhoeven AJ, Reuingsperger CP, Roos D. Human neutrophils lose their surface Fc gamma RIII ａnd acquire

Annexin V binding sites during apoptosis in vitro. Blood. 1995; 85（2）:532-540. （Biology）

Koopman G, Reuingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH. Annexin V for flow cytometric detection of phosphatidylserine expression on

B cells undergoing apoptosis. Blood. 1994; 84（5）:1415-1420. （Biology）

Martin SJ, Reuingsperger CP, McGahon AJ, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of

the initiating stimulus: inhibition by overexpression of Bcl-2 ａnd Abl. J Exp Med. 1995; 182（5）:1545-1556. （Biology）

Raynal P, Pollard HB. Annexins: the problem of assessing the biological role for a gene family of multifunctional calcium- ａnd phospholipid-binding proteins.

Biochim Biophys Acta. 1994; 1197（1）:63-93. （Biology）

van Engeland M, Ramaekers FC, Schutte B, Reuingsperger CP. A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent

cells in culture. Cytometry. 1996; 24（2）:131-139. （Biology）

Vermes I, Haanen C, Steffens-Nakken H, Reuingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early

apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods. 1995; 184（1）:39-51. （Biology）

559763