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 FITC.

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, FITC Annexin V staining can identify apoptosis at an earlier

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

FITC 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 FITC Annexin V is typically used in conjunction with a vital dye such as propidium

iodide （PI） or 7-Amino-Actinomycin （7-AAD） to allow the investigator to identify early apoptotic cells （PI negative, FITC Annexin V

positive）. Viable cells with intact membranes exclude PI, wheras the membranes of dead ａnd damaged cells are permeable to PI. For example,

cells that are considered viable are FITC Annexin V ａnd PI negative; cells that are in early apoptosis are FITC Annexin V positive ａnd PI

negative; ａnd cells that are in late apoptosis or already dead are are both FITC Annexin V ａnd PI 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 FITC Annexin V ａnd PI. However, when apoptosis is measured over time, cells can be often tracked from FITC

Annexin V ａnd PI negative （viable, or no measurable apoptosis）, to FITC Annexin V positive ａnd PI negative （early apoptosis, membrane

integrity is present） ａnd finally to FITC Annexin V ａnd PI 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 FITC Annexin V ａnd PI positive, in of itself, reveals

less information about the process by which the cells underwent their demise.

Preparation ａnd Storage

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

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Flow Cytometric Analysis of FITC Annexin V staining. Jurkat cells

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

panels） or treated for 4 hours with 12 μM campotothecin （bottom

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

containing propidium iodide （PI） ａnd analyzed by flow cytometry.

Untreated cells were primarily FITC Annexin V ａnd PI 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 （FITC Annexin V ａnd PI negative） ａnd cells undergoing

apoptosis （FITC Annexin V positive ａnd PI negative）. A minor

population of cells were observed to be FITC Annexin V ａnd PI

positive, indicating that they were in end stage apoptosis or already

dead.

Application Notes

Application

Flow cytometry Routinely Tested

Recommended Assay Procedure:

FITC 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. FITC Annexin V binding is calcium dependent ａnd defined calcium

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

FITC 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 FITC 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 FITC Annexin V Staining Protocol to measure apoptosis.

FITC ANNEXIN V STAINING PROTOCOL

FITC 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. Propidium Iodide （PI） is a standard flow cytometric viability probe ａnd is used to distinguish viable from

nonviable cells. Viable cells with intact membranes exclude PI, whereas the membranes of dead ａnd damaged cells are permeable to PI. Cells that

stain positive for FITC Annexin V ａnd negative for PI are undergoing apoptosis. Cells that stain positive for both FITC Annexin V ａnd PI are

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

alive ａnd not undergoing measurable apoptosis.

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Reagents

1. FITC Annexin V （component no. 51-65874X）: Use 5 μl per test.

2. Propidium Iodide （PI） （component no. 51-66211E） 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 FITC Annexin V ａnd 5 μl PI.

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 FITC Annexin V （no PI）.

3. Cells stained with PI （no FITC 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 FITC Annexin V and/or FITC

Annexin V ａnd PI. 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 （FITC Annexin V

positive, PI negative or FITC Annexin V positive, PI 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 PI as well as for FITC 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 FITC

Annexin V ａnd PI.

Product Notices

1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.

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.

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