BD Pharmingen™ Cy™5 Annexin V
(RUO)
Preparation And Storage
Recommended Assay Procedures
Cy™5 Annexin V is a sensitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces (Kd of ~5 x 10e2) with a higher affinity for phosphatidylserine (PS) than most other phospholipids. Cy™5 Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the Cy™5 Annexin V Staining Protocol. Investigators should note that Cy™5 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 Cy™5 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 10e6 Jurkat cells.
2. Incubate the cells for 4-6 hr at 37°C.
3. Proceed with the Cy™5 Annexin V Staining Protocol to measure apoptosis.
Cy™5 ANNEXIN V STAINING PROTOCOL
Reagents
1. Cy™5 Annexin V: Included. Use 5 µl per test.
2. 7-Amino-Actinomycin D (7-AAD): Not included. 7-AAD (Cat.No. 559925) is a convenient, ready-to-use nucleic acid dye with fluorescence detectable in the far red range of the spectrum. Use 5 µl per test.
3. 10X Binding Buffer: Not Included. 0.1 M Hepes (pH 7.4) 1.4 M NaCl, 25 mM CaCl2. Store at 4°C. Alternatively, Annexin V Binding Buffer, 10X concentrate (Cat. No. 556454) may be purchased.
Staining
1. Wash cells twice with cold PBS and then resuspend cells in 1X Binding Buffer at a concentration of 1 x 10e6 cells/ml.
2. Transfer 100 µl of the solution (1 x 10e5 cells) to a 5 ml culture tube.
3. Add 5 µl of Cy™5 Annexin V (for one and two color analysis) and 5 µl of 7-AAD (for two color analysis only).
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
Cy™5 emission wavelength is 670 nm and excitation is 625-650 nm. Cy5 is optimized for FL4 fluorescence on the BD FACSCalibur™ flow cytometer. For the BD FACSVantage™ flow cytometer, the recommended filter for emission is 675/20.
The following controls are used to set up compensation and quadrants:
1. Unstained cells.
2. Cells stained with Cy™5 Annexin V alone (no 7-AAD).
3. Cells stained with 7-AAD alone (no Cy™5 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 Cy™5 Annexin V and/or Cy™5 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 (Cy™5 Annexin V positive, 7-AAD negative or Cy™5 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 Cy™5 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 Cy™5 Annexin V and 7-AAD.
Product Notices
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- 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.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Cy is a trademark of GE Healthcare.
- Before staining with this reagent, please confirm that your flow cytometer is capable of exciting the fluorochrome and discriminating the resulting fluorescence.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
Companion Products
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 Cy™5. 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, Cy™5 Annexin V staining can identify apoptosis at an earlier stage than assays based on nuclear changes such as DNA fragmentation.
Cy™5 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 Cy™5 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 (7-AAD negative, Cy™5 Annexin V positive). Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to 7-AAD. For example, cells that are considered viable are both Cy™5 Annexin V and 7-AAD negative while cells that are in early apoptosis are Cy™5 Annexin V positive and 7-AAD negative, while cells that are in late apoptosis or already dead are both Cy™5 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 Cy™5 Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from Cy™5 Annexin V and 7-AAD negative (viable, or no measurable apoptosis), to Cy™5 Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present) and finally to Cy™5 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 Cy™5 Annexin V and 7-AAD positive, in of itself, reveals less information about the process by which the cells underwent their demise.
Development References (7)
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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. (Methodology: Apoptosis, Flow cytometry). View Reference
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Homburg CH, de Haas M, von dem Borne AE, Verhoeven AJ, Reutelingsperger 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: Apoptosis). View Reference
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Koopman G, Reutelingsperger 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. (Methodology: Apoptosis, Flow cytometry). View Reference
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Martin SJ, Reutelingsperger 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: Apoptosis). View Reference
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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). View Reference
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Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger 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. (Methodology: Apoptosis, Flow cytometry). View Reference
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van Engeland M, Ramaekers FC, Schutte B, Reutelingsperger CP. A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry. 1996; 24(2):131-139. (Methodology: Apoptosis, Flow cytometry). View Reference
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Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
