Annexin V-FITC Apoptosis Detection Kit
(Store at 4°C; Stable for one year)
I. Introduction
Apoptosis is a normal physiologic process which occurs during embryonic development as well as in maintenance 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. In apoptotic cells, the membrane phospholipids 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 such as 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
The Annexin V-FITC Apoptosis Detection Kit is based on the observation that soon after initiating apoptosis, most cell types translocate the membrane phospholipids phosphatidylserine (PS) from the inner face of the plasma membrane to the cell surface. Once on the cell surface, PS can be easily detected by staining with a fluorescent (FITC) conjugate of annexin V. The one-step staining procedure takes only 20 minutes. In addition, the assay can be directly performed on live cells, without the need for fixation. Detection can be analyzed by flow cytometry or by fluorescence microscopy.
II. Kit Contents
Components | B620011 25 assays | B620012 50 assays | B620013 100 assays | B620014 400 assays |
Annexin V-FITC | 125μL | 250μL | 500μL | 2 mL |
1X Binding Buffer | 10 mL | 20 mL | 40mL | 160mL |
Propidium Iodide | 250μL | 500μL | 1 mL | 4 mL |
III. Annexin V-FITC Assay Protocol
A. Incubation of cells with Annexin V-FITC
1. Wash cells twice with cold PBS, gently remove the PBS from cell pellet.
2. Resuspend cells in 400μl with 1X binding buffer at a concentration of 1 X 106 cells/ml.
3. Add 5 µl of Annexin V�FITC and gently vortex the cells and incubate for 15 min at 4-8°C in the dark.
4. Add 10 µl of PI to tube for another 5 min at 4-8°C in the dark.
5. Analyze by flow cytometry or by Fluorescence Microscopy as soon as possible (within 1 hr).
B. Quantification by Flow Cytometry
1. Generate a line FSC vs line SSC dot plot, run the control tube of unstained cells and gate the interested population.
2. Generate a log FL1 vs log FL2 (or FL3 the best) dot plot of the gated cells. Adjust parameters so that most of events are located within the lower left quadrant and are within the first log decade on both the X and Y axis of the FL1 vs FL2 (or FL3) dot plot.
3. Run only Annexin V-FITC staining tube. Adjust the compensation ensure that no events are recorded in the upper left and upper right quadrants of the display. This could achieve by increasing FL2 (or FL3) - %FL1 compensation (range from 15-25%).
4. Run only PI staining tube and adjust the compensation to ensure that no events are recorded in the upper and lower right quadrants of the display. Decrease the FL1 - %FL2 (or FL3) compensation (may range from 0-1%).
5. Run samples and collect over 10,000 cells in the mode.
C. Detection by Fluorescence Microscopy
1. Place the cell suspension after Step A.4 on a glass slide. Cover the cells with a glass coverslip.
For analyzing adherent cells, grow cells directly on a coverslip. Following incubation (after Step A.4), invert coverslip on a glass slide and visualize cells. The cells can also be washed and fixed in 2% formaldehyde before visualization. (Cells must be incubated with Annexin V-FITC before fixation since any cell membrane disruption can cause nonspecific binding of Annexin V to PS on the inner surface of the cell membrane.)
2. Observe the cells under a fluorescence microscope using a dual filter set for FITC.
Cells that have bound Annexin V-FITC will show green staining in the plasma membrane. Cells that have lost membrane integrity will show red staining (PI) throughout the nucleus and a halo of green staining (FITC) on the cell surface (plasma membrane).
IV. Note
1. All chemicals should be considered as potentially hazardous. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses and gloves. Care should be taken to avoid contact with skin or eyes. In the case of contact with skin or eyes wash immediately with water. See material safety data sheet(s) and/or safety statements(s) for specific advice.
2. Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures.
3. Do not mix or substitute reagents with those from other lots or other sources.
4. Do not use kit reagents beyond expiration date on label.
5. Do not expose kit reagents to strong light during storage or incubation.
6. Do not pipette by mouth.
7. Do not eat or smoke in areas where kit reagents or samples are handled.
8. Avoid contact of skin or mucous membranes with kit reagents.
9. Rubber or disposable latex gloves should be worn while handling kit reagents.
10. Avoid splashing or generation of aerosols.
11. In order to avoid microbial contamination or cross-contamination of reagents which may invalidate the test use disposable pipette tips and/or pipettes.
12. Glass-distilled water or deionized water must be used for reagent preparation.
13. Decontaminate and dispose cells and all potentially contaminated materials as if they could contain infectious agents. The preferred method of decontamination is autoclaving for a minimum of 1 hour at 121.5°C.
14. Liquid wastes not containing acid and neutralized waste may be mixed with sodium hypochlorite in volumes such that the final mixture contains 1.0% sodium hypochlorite. Allow 30 minutes for effective decontamination. Liquid waste containing acid must be neutralized prior to the addition of sodium hypochlorite.
V. Limitations
1. Bacterial or fungal contamination of either screen samples or reagents or cross-contamination between reagents may cause erroneous results.
2. Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergent before use.
VI. Related Products
B620031 | Annexin V EGFP Apoptosis Detection kit | 25 assays |
B620032 | Annexin V EGFP Apoptosis Detection kit | 50 assays |
B620033 | Annexin V EGFP Apoptosis Detection kit | 100 assays |
B620034 | Annexin V EGFP Apoptosis Detection kit | 400 assays |
B620021 | Annexin V PE Apoptosis Detection kit | 25 assays |
B620022 | Annexin V PE Apoptosis Detection kit | 50 assays |
B620023 | Annexin V PE Apoptosis Detection kit | 100 assays |
B620024 | Annexin V PE Apoptosis Detection kit | 400 assays |