Rapid, clear assessment of apoptosis

Annexin V conjugates are useful for detecting translocated phosphatidylserine (PS), a hallmark of apoptosis. Annexin V conjugates can be used in combination with other dyes, including nucleic acid stains, to accurately assess mixed populations of apoptotic and nonapoptotic cells. We offer annexin V conjugates as stand alone reagents or easy-to-use kits.

Flow cytometric detection of apoptosis with annexin V conjugates

We have collaborated with Nexins Research BV—the original developer of fluorescent phosphatidylserine-binding proteins—to produce annexin V conjugates with superior brightness. These annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine, an indicator of intermediate stages of apoptosis. 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.

The benefits of our annexin V conjugates include:

  • Conjugated to Alexa Fluor™ dyes for brighter signals
  • Conjugates for all available lasers for increased multiplexing capabilities
  • Available as stand-alone reagents or easy-to-use kits

Annexin V reagent selection guides

Annexin V conjugates for flow cytometry (standalone)

Conjugate Laser Emission Recommended filter Size
Annexin V, Alexa Fluor™ 350 UV 442 nm 450/40 nm 100 assays
Annexin V, Pacific Blue™ 405/7 nm 455 nm 450/50 nm 100 assays
Annexin V, Alexa Fluor 488 488 nm 519 nm 530/30 nm 100 assays
Annexin V, Fluorescein 488 nm 518 nm 530/30 nm 100 assays
Annexin V, RPE 488 nm 578 nm 585/42 nm 50 assays
Annexin V, Alexa Fluor 555 532 nm 565 nm 575/26 nm 100 assays
Annexin V, Alexa Fluor 568 561 nm 603 nm 610/20 nm 100 assays
Annexin V, Alexa Fluor 594 590 nm 617 nm 630/20 nm 100 assays
Annexin V, APC 633/5 nm 665 nm 661/8 nm 100 assays
Annexin V, Alexa Fluor 647 633/5 nm 660 nm 661/8 nm 50 assays
Annexin V, biotin-X conjugate NA NA NA 100 assays
Required Buffer
Annexin Binding Buffer (5x)

Dead cell stain kits with Annexin V conjugates

Annexin V conjugate Dead cell stain Approximate fluorescence excitation/emission maxima Additional reagents in kit Size Cat. No.
Annexin V conjugate Dead cell stain
Annexin V, Alexa Fluor™ 488  PI 499/521 nm 535/617 nm
  • Annexin binding buffer (5x)
50 assays V13241
250 assays V13245
Annexin V, Fluorescein PI 494/518 nm 535/617 nm 50 assays V13242
Annexin V, APC  SYTOX™ Green 650/660 nm 503/524 nm 50 assays V35113
Annexin V, Pacific Blue™ SYTOX™ AADvanced™ 415/455 nm 546/647 nm 50 assays A35136
Annexin V, RPE  SYTOX Green 488/575 nm 503/524 nm 50 assays V35112
Annexin V, Alexa Fluor 488 SYTOX Green 499/521 nm 503/524 nm 50 assays V13240
Annexin V, APC  SYTOX Green 650/660 nm 503/524 nm
  • Annexin binding buffer (5x)
  • C12-resazurin (571/585 nm)
50 assays V35114

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. Prepare annexin-binding buffer: 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4.
  2. Induce apoptosis in cells using the desired method. A negative control should be prepared by incubating cells in the absence of inducing agent.
  3. Harvest the cells after the incubation period and wash in cold phosphate-buffered saline (PBS).
  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.
  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.
  6. Incubate the cells at room temperature for 15 minutes.
  7. After the incubation period, add 400 μL of annexin-binding buffer, mix gently, and keep the samples on ice.
  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 secondary detection 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.

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 to identify apoptotic cells and with propidium iodide 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 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  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 PE Conjugate.
Jurkat cells were induced with 10 µM camptothecin, then cells were washed in annexin-binding buffer and stained with Annexin V PE and SYTOX™ Green. Cells were analyzed using flow cytometry with 488 nm excitation.