Description
Alexa Fluor™ 647 Annexin V is a sensitive probe for identifying apoptotic cells, binding to negatively charged phospholipid surfaces with a higher affinity for phosphatidylserine (PS) than most other phospholipids. Alexa Fluor™ 647 Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the Alexa Fluor™ 647 Annexin V Staining Protocol. Investigators should note that Alexa Fluor™ 647 Annexin V flow cytometric analysis on adherent cell types (eg, 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 Engeland et al.). INDUCTION OF APOPTOSIS BY CAMPTOTHECIN The following protocol is provided as an illustration on how Alexa Fluor™ 647 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 × 10^6 Jurkat cells. 2. Incubate the cells for 4-6 hr at 37°C. 3. Proceed with the Alexa Fluor™ 647 Annexin V Staining Protocol to measure apoptosis. Reagents 1. Alexa Fluor™ 647 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 Annexin Binding Buffer: Not Included. 0.1 M Hepes (pH 7.4) 1.4 M NaCl, 25 mM CaCl2. Store at 4°C. Alternatively, BD Pharmingen™ Annexin V Binding Buffer, 10X concentrate (Cat. No. 556454) may be purchased. Alexa Fluor™ 647 ANNEXIN V STAINING PROTOCOL Staining 1. Wash cells twice with cold PBS and then resuspend cells in 1× Binding Buffer at a concentration of 1 × 10^6 cells/ml. 2. Transfer 100 µl of the cell suspension (1 × 10^5 cells) to a 5 ml culture tube. 3. Add 5 µl of Alexa Fluor™ 647 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 1× Annexin V 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 Alexa Fluor™ 647 Annexin V alone (no 7-AAD). 3. Cells stained with 7-AAD alone (no Alexa Fluor™ 647 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 Alexa Fluor™ 647 Annexin V alone or with Alexa Fluor™ 647 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 (Alexa Fluor™ 647 Annexin V positive, 7-AAD negative or Alexa Fluor™ 647 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 the 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 Alexa Fluor™ 647 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 Alexa Fluor™ 647 Annexin V and 7-AAD.
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions and unreacted dye was removed.
1. Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
2. Alexa Fluor® 647 fluorochrome emission is collected at the same instrument settings as for allophycocyanin (APC).
3. 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.
4. 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).
5. For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
6. Source of all serum proteins is from USDA inspected abattoirs located in the United States.
7. This product is provided under an intellectual property license between Life Technologies Corporation and BD Businesses. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. For information on purchasing a license to this product for any other use, contact Life Technologies Corporation, Cell Analysis Business Unit Business Development, 29851 Willow Creek Road, Eugene, OR 97402, USA, Tel: (541) 465-8300. Fax: (541) 335-0504.
8. Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
9. Alexa Fluor™ is a trademark of Life Technologies Corporation.
10. For U.S. patents that may apply, see bd.com/patents.
