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.
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 Invitrogen Alexa Fluor and eFluor 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 conjugates for flow cytometry (standalone)
|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|
|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, eFluor 450||7-AAD||405/450 nm||NA||Annexin binding buffer (10x)||50 assays||88-8006-72|
|Annexin V, Pacific Blue||SYTOX AADvanced||415/455 nm||546/647 nm||Annexin binding buffer (5x)||50 assays||A35136|
|Annexin V, Alexa Fluor 488||PI||499/521 nm||535/617 nm||
Annexin binding buffer (5x)
|Annexin V, Alexa Fluor 488||SYTOX Green||499/521 nm||503/524 nm||Annexin binding buffer (5x)||50 assays||V13240|
|Annexin V, Fluorescein||PI||494/518 nm||535/617 nm||Annexin binding buffer (5x)||50 assays||V13242|
|Annexin V, PerCP-eFluor 710||—||482/710 nm||NA||Annexin binding buffer (10x)||50 assays||88-8008-72|
|Annexin V, APC||SYTOX Green||650/660 nm||503/524 nm||Annexin binding buffer (5x)||50 assays||V35113|
|Annexin V, RPE||SYTOX Green||488/575 nm||503/524 nm||Annexin binding buffer (5x)||50 assays||V35112|
|Annexin V, RPE-Cyanine7||—||488/767 nm||NA||Annexin binding buffer (10x)||50 assays||88-8103-72|
|Annexin V, APC||SYTOX Green||650/660 nm||503/524 nm||
--Annexin binding buffer (5x)
--C12-resazurin (571/585 nm)
Figure 1. 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.
Figure 2. 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.
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.
- Prepare annexin-binding buffer: 10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl2, pH 7.4.
- Induce apoptosis in cells using the desired method. A negative control should be prepared by incubating cells in the absence of inducing agent.
- Harvest the cells after the incubation period and wash in cold phosphate-buffered saline (PBS).
- 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.
- Add 5 μL of the annexin V conjugate to each 100 μL of cell suspension. You may also wish to add an appropriate Invitrogen dead-cell indicator such as propidium iodide, SYTOX Green dye, or SYTOX AADvanced dead cell stain.
- Incubate the cells at room temperature for 15 minutes.
- After the incubation period, add 400 μL of annexin-binding buffer, mix gently, and keep the samples on ice.
- 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.
For Research Use Only. Not for use in diagnostic procedures.