Description
Apoptosis is a normal physiologic process that 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. Loss of plasma membrane asymmetry 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 BD Horizon™ PE-CF594. 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-CF594 Annexin V staining can identify cells undergoing apoptosis at an earlier stage rather than assays based on nuclear changes such as DNA fragmentation.
PE-CF594 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-CF594 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 (PE-CF594 Annexin V positive, 7-AAD negative). Viable cells with intact membranes exclude 7-AAD, whereas the membranes of dead and damaged cells are permeable to the nucleic acid dye, 7-AAD.For example, cells that are considered viable are both PE-CF594 Annexin V negative and 7-AAD negative while cells that are in early apoptosis are PE-CF594 Annexin V positive and 7-AAD negative. Cells that are in late apoptosis or already dead are both PE-CF594 Annexin V positive 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-CF594 Annexin V and 7-AAD. However, when apoptosis is measured over time, cells can be often tracked from being PE-CF594 Annexin V negative and 7-AAD negative (viable, or no measurable apoptosis), to PE-CF594 Annexin V positive and 7-AAD negative (early apoptosis, membrane integrity is present), and finally to PE-CF594 Annexin V positive 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-CF594 Annexin V and 7-AAD positive, by itself reveals less information about the process by which the cells underwent their demise.
The Annexin V is conjugated to BD Horizon PE-CF594 that has been developed for use in multicolor flow cytometry experiments and is available exclusively from BD Biosciences. Annexin is conjugated to BD Horizon PE-CF594, which has been developed exclusively by BD Biosciences as a better alternative to PE-Texas Red®. PE-CF594 excites and emits at similar wavelengths to PE-Texas Red® yet exhibits improved brightness and spectral characteristics. Due to PE having maximal absorption peaks at 496 nm and 564 nm, PE-CF594 can be excited by the blue (488-nm), green (532-nm) and yellow-green (561-nm) lasers and can be detected with the same filter set as PE-Texas Red® (eg, 610/20-nm filter).
When compensating dyes in this spectral range, the most accurate compensation can be obtained using unstained and single color-stained cellular controls.
Format
- FormatPE-CF594
- Excitation SourceBlue 488 nm,Green 532 nm,Yellow/Green 561 nm
- Excitation Max496nm
- Emission Max612nm
BD Horizon™ PE-CF594 is a tandem conjugate, developed exclusively by BD Biosciences, that combines PE and CF594. PE-CF594 is a brighter alternative to PE-Texas Red®, with improved spectral characteristics. PE-CF594 reagents exhibit very consistent spillover values lot to lot, making them an ideal choice for the PE-Texas Red® detector (610/20 nm).
Other Formats→
- APC
- BUV395
- BV421
- BV605
- BV711
- Biotin
- Cy5
- Cy5.5
- FITC
- PE
- PerCP-Cy™5.5
- Purified
- V450
- V500
Suggested Companion Products
Stain Buffer (FBS) RUO 500mLCat No: 554656
Annexin V Binding Buffer, 10X concentrate RUO 50mLCat No: 556454
7-AAD RUO 2mLCat No: 559925
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Preparation and Storage
Store undiluted at 4°C and protected from prolonged exposure to light.Do not freeze.
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).
- 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.
- Source of all serum proteins is from USDA inspected abattoirs located in the United States.
- Please observe the following precautions:Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
- When excited by the yellow-green (561-nm) laser, the fluorescence may be brighter than when excited by the blue (488-nm) laser.
- Because of the broad absorption spectrum of the tandem fluorochrome, extra care must be taken when using multi-laser cytometers, which may directly excite both PE and CF™594.
- This product is provided under an Agreement between BIOTIUM and BD Biosciences. The manufacture, use, sale, offer for sale, or import of this product is subject to one or more patents or pending applications owned or licensed by Biotium, Inc.This product, and only in the amount purchased by buyer, may be used solely for buyer’s own internal research, in a manner consistent with the accompanying product literature.No other right to use, sell or otherwise transfer (a) this product, or (b) its components is hereby granted expressly, by implication or by estoppel.This product is for research use only.Diagnostic uses require a separate license from Biotium, Inc. For information on purchasing a license to this product including for purposes other than research, contact Biotium, Inc., 3159 Corporate Place, Hayward, CA 94545, Tel: (510) 265-1027.Fax: (510) 265-1352. Email: btinfo@biotium.com.
- Texas Red is a registered trademark of Molecular Probes, Inc., Eugene, OR.
- CF™ is a trademark of Biotium, Inc.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
BD Horizon PE-CF594 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. PE-CF594 Annexin V binding is calcium dependent and defined calcium and salt concentrations are required for optimal staining as described in the PE-CF594 Annexin V Staining Protocol. Investigators should note that PE-CF594 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 Engelend et al.).
INDUCTION OF APOPTOSIS BY CAMPTOTHECIN
The following protocol is provided as an illustration on how PE-CF594 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 PE-CF594 Annexin V Staining Protocol to measure apoptosis.
Reagents
1. BD Horizon PE-CF594 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.
BD Horizon PE-CF594 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 PE-CF594 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 PE-CF594 Annexin V alone (no 7-AAD).
3. Cells stained with 7-AAD alone (no PE-CF594 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-CF594 Annexin V alone or with PE-CF594 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-CF594 Annexin V positive, 7-AAD negative or PE-CF594 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 PE-CF594 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-CF594 Annexin V and 7-AAD.
