Annexin V Conjugates for Flow CytometryAnnexin V conjugates are very useful for flow cytometry and confocal or epifluorescence microscopy and, like antibody staining, can be used in combination with other dyes, including nucleic acid stains, to accurately assess mixed populations of apoptotic and nonapoptotic cells.

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Apoptosis Detection with Annexin V Conjugates

Our annexin V conjugates and assays offer:
  • Sensitivity—conjugated to Alexa Fluor® dyes for brighter signals
  • Flexibility—conjugates for many lasers and detection channels
  • Simplicity—assay procedure typically completed in less than 30 minutes

The Best and Brightest Annexin V Conjugates Available
Molecular Probes is collaborating with Nexins Research BV—the original developer of fluorescent phosphatidylserine-binding proteins—to provide what we feel are the best and brightest annexin V conjugates available. The human vascular anticoagulant annexin V is a 35–36 kDa, Ca2+-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine (PS). In normal viable cells, PS is located on the cytoplasmic surface of the cell membrane. However, in apoptotic cells, PS is translocated from the inner to the outer leaflet of the plasma membrane, exposing PS to the external cellular environment where it can be detected by annexin V conjugates. In leukocyte apoptosis, PS on the outer surface of the cell marks the cell for recognition and phagocytosis by macrophages.

Annexin V Conjugates for Flow Cytometry
Highly fluorescent annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine, an indicator of intermediate stages of apoptosis. Nuclear fragmentation, mitochondrial membrane potential flux, and caspase-3 activation apparently precede phosphatidylserine "flipping" during apoptosis, whereas permeability to propidium iodide and cytoskeletal collapse occur later. The difference in fluorescence intensity between apoptotic and nonapoptotic cells stained with our fluorescent annexin V conjugates, as measured by flow cytometry, is typically about 100-fold.

Annexin V conjugates are very useful for flow cytometry and confocal or epifluorescence microscopy and, like antibody staining, can be used in combination with other dyes, including nucleic acid stains, to accurately assess mixed populations of apoptotic and nonapoptotic cells. Our annexin V conjugates are available as stand-alone reagents, each suitable for 50–100 flow cytometry assays, many more imaging assays, or in several variations of our multiparametric apoptosis assays for flow cytometry.
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Annexin V Reagent Selection Guide

 Conjugate  Laser Emission Recommended filter Size Cat. No. 
Annexin V Alexa Fluor® 350UV442 nm450/40 nm100 assaysA23202
Annexin V Pacific Blue™405/7 nm455 nm450/50 nm100 assaysA35122
Annexin V Alexa Fluor® 488488 nm519 nm530/30 nm100 assaysA13201
Annexin V RPE488 nm578 nm585/42 nm50 assaysA35111
Annexin V Alexa Fluor® 555532 nm565 nm575/26 nm100 assaysA35108
Annexin V Alexa Fluor® 568561 nm603 nm610/20 nm100 assaysA13202
Annexin V Alexa Fluor® 594590 nm617 nm630/20 nm100 assaysA13203
Annexin V APC633/5 nm665 nm661/8 nm100 assaysA35110
Annexin V Alexa Fluor® 647633/5 nm660 nm661/8 nm50 assaysA23204

 

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Staining Protocol

We have optimized this assay using Jurkat cells treated with camptothecin to induce apoptosis. Some modifications may be required or use with other cell types.

1.1 Prepare annexin-binding buffer: 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4.

1.2 Induce apoptosis in cells using the desired method. A negative control should be prepared by incubating cells in the absence of inducing agent.1.3 Harvest the cells after the incubation period and wash in cold phosphate-buffered saline (PBS).

1.4 Pellet the washed cells, discard the supernatants, and resuspend the cells in annexin-binding buffer. Determine the cell density and dilute in annexin-binding buffer to ~1 × 106 cells/mL, preparing a sufficient volume to have 100 μL per assay.

1.5 Add 5 μL of the annexin V conjugate to each 100 μL of cell suspension. You may also wish to add an appropriate dead-cell indicator such as propidium iodide, SYTOX® Green dye, or SYTOX® AADvanced™ dead cell stain.

1.6 Incubate the cells at room temperature for 15 minutes.

1.7 After the incubation period, add 400 μL of annexin-binding buffer, mix gently, and keep the samples on ice.

1.8 As soon as possible, analyze the stained cells by flow cytometry. Cells labeled with the biotin-X conjugate of annexin V will require the application of a secondarydetection agent such as fluorophore-labeled streptavidin. The population should separate into at least two groups: live cells with only a low level of fluorescence, and apoptotic cells that exhibit substantially higher fluorescence intensity. If a dead-cell stain is used, dead cells will be labeled with both the dead-cell stain and the annexin V conjugate.

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Supporting Annexin V Journal Citations

Annexin V CitationsAlexa Fluor® 488
  • Nelson Arispe et al. (2004) Hsc70 and Hsp70 interact with phosphatidylserine onthe surface of PC12 cells resulting in a decrease of viability. FASEB 18:1636–1645.
  • James E. Kirby  (2004) Anthrax lethal toxin induces human endothelial cell apoptosis. Infection and Immunity 72:430–439.
  • Teresa Puig et al. (2008) Fatty acid metabolism in breast cancer cells: differential inhibitory effects of epigallocatechin gallate (EGCG) and C75. Breast Cancer Research and Treatment 109:471–479.

Pacific Blue™
  • Timothy H Witney et al. (2009) A comparison between radiolabeled fluorodeoxyglucose uptake and hyperpolarized 13C-labeled pyruvate utilization as methods for detecting tumor response to treatment. Neoplasia 11: 574–582.
  • Rachel L. Zemans et al. (2009) A novel method for long term bone marrow culture and genetic modification of murine neutrophils via retroviral transduction. Journal of Immunological Methods 340:102–115.

Alexa Fluor® 350
  • Douglas L. Miller and Chunyan Dou (2009) Induction of apoptosis in sonoporation and ultrasonic gene transfer. Ultrasound Med Biology 35:144–154.

Alexa Fluor® 647 and APC
  • Partha Mukhopadhyay et al. (2007) Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy. Nature Protocols 2:2295–2301.

Alexa Fluor® 647
  • Hajime Hiraragi et al. (2005) Human T-lymphotropic virus type 1 mitochondrion-localizing protein p13ii sensitizes Jurkat T cells to Ras-mediated apoptosis. Journal of Virology 79:9449–9457.
  • Leonarda Troiano et al. (2007) Multiparametric analysis of cells with different mitochondrial membrane potential during apoptosis by polychromatic flow cytometry. Nature Protocols 2:2719–2727.

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See Our Annexin V Conjugates in Action

Annexin V Alexa Fluor® 488 Conjugate
Jurkat cells (T cell leukemia, human) treated with 10 μM camptothecin for 4 hours (right panel) or untreated (as control, left panel). Cells were then treated with Annexin V Alexa Fluor® 488 (Cat. No. A13201) to identify apoptotic cells and with propidium iodide (Cat. No. P3566) to identify dead cells, followed by flow cytometric analysis. Note that the camptothecin-treated cells (right panel) have a higher percentage of apoptotic cells (indicated by an “A”) than the basal level of apoptosis seen in the control cells (left panel). V = viable cells, D = dead cells.

Annexin V Alexa Fluor® 488 Conjugate
Jurkat human T cell leukemia cells were treated with 1 μM camptothecin. The externalized phosphatidylserine, a characteristic of early-stage apoptotic cells, was detected with Annexin V Alexa Fluor® 488 (Cat. No. A13201). The late-stage apoptotic and necrotic cells were stained with propidium iodide (Cat. No. P3566). The image was acquired using bandpass filters appropriate for fluorescein.

Annexin V Alexa Fluor® 647 Conjugate
Jurkat cells were induced with 10 µM camptothecin, then cells were washed and stained with Annexin V Alexa Fluor® 647 conjugate (Cat. No. A23204) and SYTOX® Green nucleic acid stain (Cat. No S7020). Cells were analyzed by flow cytometry using 488 nm and 633 nm excitation.

Annexin V Alexa Fluor® 568 Conjugate
Jurkat cells were induced with 10µM camptothecin, then cells were washed and stained with Annexin V Alexa Fluor® 568 conjugate (Cat. No. A13202) and propidium iodide (Cat. No. P3566). Cells were analyzed using flow cytometry with 488 nm and 532 nm excitation and 585⁄42 nm emission and 610⁄20 nm bandpass filters.

Annexin V Alexa Fluor® 350 Conjugate
Jurkat cells were induced with 10 µM camptothecin, then washed and stained with Annexin V Alexa Fluor® 350 conjugate (Cat. No. A23202) and propidium iodide (Cat. No. P3566). Cells were analyzed using flow cytometry with 365 nm and 488 nm excitation.

Annexin V Pacific Blue™ Conjugate
Jurkat cells (T cell leukemia, human) were treated with 10 µM camptothecin for 4 hours. Cells were then treated with the reagents in the Pacific Blue™ Annexin V⁄SYTOX® AADvanced™ Apoptosis Kit (Cat. No. A35136) and analyzed by flow cytometry using 405 nm and 488 nm excitation. Note that the camptothecin-treated cells have a higher percentage of apoptotic cells (1B) than the basal level of apoptosis seen in the control cells (1A). A = apoptotic cells, V = viable cells, N = necrotic cells.

Annexin V APC Conjugate
Jurkat cells were induced with 10 µM camptothecin, then cells were washed in annexin-binding buffer and stained with Annexin V APC (Cat. No. A35110) and SYTOX® Green (Cat. No. S7020). Cells were analyzed using flow cytometry with 488 nm and 633 nm excitation sources.

Annexin V PE Conjugate
Jurkat cells were induced with 10 µM camptothecin, then cells were washed in annexin-binding buffer and stained with Annexin V PE (Cat. No. A35111) and SYTOX® Green (Cat. No. S7020). Cells were analyzed using flow cytometry with 488 nm excitation.

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