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Annexin-V Staining

Alterations in the plasma membrane are a hallmark indicator of early-stage apoptosis in living cells. During the initial phases of apoptosis, phosphatidylserine (PS) residues translocate from the inner to the outer leaflet of the plasma membrane. In cell imaging and flow cytometry, fluorescently labeled annexin V conjugates are commonly used to detect apoptotic cells by their ability to bind to externalized PS residues.

 



Annexin V


Annexins are a family of calcium-dependent phospholipid-binding proteins. They are found in abundance in eukaryotic organisms (animal, plant, and fungi), where they play critical roles in various cellular and physiological processes such as signal transduction, vesicle transport, and membrane scaffolding.

Annexin V is a 35 kDa phospholipid-binding protein. Its strong calcium-dependent affinity for phosphatidylserine (PS) can be used to identify cells undergoing apoptosis. In viable cells, negatively charged PS residues are located on the cytosolic surface of the plasma membrane. However, during early apoptosis, cells lose plasma membrane asymmetry, and PS residues translocate to the outer leaflet of the membrane, where fluorescently labeled annexin V conjugates can detect them.

 

Table 1. Overview of annexin V conjugates.

Specification
Value
ApplicationDetects externalized phosphatidylserine to identify apoptotic cells
ReadoutFluorescence microscopy, live cell imaging and flow cytometery
Live CellsYes
Fixed CellsNot for use
FixableYes, fix after staining with formaldehyde
MultiplexingYes, bright signals and broad wavelength ranges for multiplex analysis

 

Annexin V Conjugates for Detecting Apoptosis



Jurkat cells were treated with 1 µM staurosporine for 4 hours to induce apoptosis. Following treatment, cells were stained with Annexin V-iFluor™ 555 conjugate (Cat No. 20072). The nuclei of dead cells were labeled using Nuclear Green™ DCS1 (Cat No. 17550). Images were acquired on a confocal microscope.
Annexin V conjugated to iFluor™ dyes provides a fast and reliable method for detecting externalized PS, a key characteristic in early-stage apoptosis. The superior brightness and photostability of Annexin V iFluor™ probes significantly outperform most Alexa Fluor® and other spectrally similar annexin v conjugates. Annexin V iFluor™ conjugates are useful for live-cell imaging, immunofluorescence, and flow cytometry. They can be combined with other dyes, such as nucleic acid stains, to accurately assess mixed populations of apoptotic, necrotic, and dead cells.

AAT Bioquest offers a wide variety of fluorescently labeled annexin V conjugates in colors ranging from UV to infrared for improved multiplexing flexibility. We also offer recombinant annexin V and easy-to-use kits optimized for flow cytometry, as well as biotinylated annexin V, which can be detected using fluorescently labeled streptavidin.

 

Table 2. Available annexin V conjugates for apoptosis detection.

Annexin V Label
Ex (nm)
Em (nm)
Filter Set
ε¹
Φ²
Unit Size
Cat No.
iFluor™ 350345450DAPI200000.95100 tests (200 µL)20070
iFluor™ 488491516FITC750000.9100 tests (200 µL)20071
iFluor™ 555557570TRITC1000000.64100 tests (200 µL)20072
iFluor™ 594588604Texas Red1800000.53100 tests (200 µL)20073
iFluor™ 633640654Texas Red2500000.29100 tests (200 µL)20069
iFluor™ 647656670Cy52500000.25100 tests (200 µL)20074
iFluor™ 680684701Cy5.52200000.23100 tests (200 µL)20075
iFluor™ 700690713Cy5.52200000.23100 tests (200 µL)20077
iFluor™ 750757779Cy72750000.12100 tests (200 µL)20076
XFD350 (Alexa Fluor® 350 equivalent)343441DAPI19000N/D³100 tests (200 µL)20090
  1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).
  3. N/D = not determined.

 

Annexin V Conjugates for Flow Cytometry



The detection of binding activity of Annexin V-mFluor™ Violet 450 conjugate to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Green) or with 1 µM staurosporine (Red) at 37°C for 4 hours, and then ed with Annexin V-mFluor™ Violet 450 conjugate for 30 minutes. Fluorescence intensity was measured using an ACEA NovoCyte flow cytometer in the Pacific Blue channel.
Annexin V conjugated to mFluor™ dyes, PacBlue, APC and PE are valuable tools for measuring apoptosis by flow cytometry. These conjugates can be combined with other dyes, such as nucleic acid stains, to accurately assess mixed populations of apoptotic, necrotic, and dead cells. We offer annexin V conjugates for flow cytometry as stand-alone reagents or easy-to-use kits.

Key features of Annexin V mFluor™ Conjugates

  • Superior brightness and improved photostability for long-term cellular imaging
  • pH-insensitive fluorescence over a wide molar range
  • Large Stokes shifts to reduce cross-talk between the excitation source and fluorescence emission for cellular imaging with high signal-to-noise ratios
  • Compatible with live cells only
  • Fixable after staining with formaldehyde
  • Conjugates are efficiently excited by common lasers for increased multiplexing capabilities

 

Table 3. Available annexin V conjugates for optimized for flow cytometry.

Annexin V Label
Laser
Ex (nm)
Em (nm)
Channel
ε¹
Φ²
Unit Size
Cat No.
PacBlueViolet Laser410455DAPI/FL-1 Channel460000.78100 tests (200 µL)20089
mFluor™ Violet 450Violet Laser403454DAPI/FL-1 Channel250000.92100 tests (200 µL)20080
mFluor™ Violet 510Violet Laser414510FITC/FL-1 Channel300000.86100 tests (200 µL)20081
mFluor™ Violet 540Violet Laser424560PE/FL-2 Channel150000.64100 tests (200 µL)20082
mFluor™ Blue 570Blue Laser533570PE/FL-2 Channel1200000.08100 tests (200 µL)20085
  1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. Φ = fluorescence quantum yield in aqueous buffer (pH 7.2).

 

Cell Meter™ Annexin V Binding Apoptosis Assays


Cell Meter™ Annexin V Binding Apoptosis Assay Kits provide a fast and convenient method for measuring apoptotic cells based on the externalization of phosphatidylserine. Each kit contains a recombinant annexin V conjugated to either an iFluor™ dye, mFluor™ dye, APC, PE, or FITC and used to detect translocated PS in cells undergoing early apoptosis. In addition, these kits (excluding 22825, 22826, and 22827) include a ready-to-use solution of propidium iodide (PI) or Nuclear Red™ DCS1 nucleic acid stain. Both PI and Nuclear Red™ DCS1 are impermeant to live cells but stain necrotic and dead cells with intense red fluorescence. After staining cells with annexin V conjugate and nucleic acid stain, apoptotic cells show annexin V conjugate fluorescence, dead cells show red and annexin V conjugate fluorescence, and live cells show little or no fluorescence. Cell populations can readily be differentiated using a flow cytometer.


The detection of binding activity of Annexin V-mFluor™ Violet 450 conjugate to phosphatidylserine in Jurkat cells. Jurkat cells were treated without (Green) or with 1 µM staurosporine (Red) at 37°C for 4 hours, and then ed with Annexin V-mFluor™ Violet 450 conjugate for 30 minutes. Fluorescence intensity was measured using an ACEA NovoCyte flow cytometer in the Pacific Blue channel.

Key features of Cell Meter™ Annexin V Binding Apoptosis Assays

  • High Sensitivity - annexin V labeled with iFluor™, or mFluor™ dyes produce significantly brighter and more photostable signals than Alexa Fluor and other spectrally similar annexin V conjugates
  • Flexibility - wide range of fluorescently labeled annexin V conjugates to match any instrument set-up
  • Convenient design - streamlined assay procedure which can be completed in 30 to 60 minutes

 

Table 4. Cell Meter™ Annexin V Binding Apoptosis Assay Selection Guide

Annexin V Conjugate
Dead Cell Stain
Annexin V Conjugate Ex/Em
Dead Cell stain Ex/Em
Unit Size
Cat No.
Annexin V-iFluor™ 488PI490/ 520 nm534/617 nm100 Tests22824
Annexin V-iFluor™ 555None554/578 nm***100 Tests22825
Annexin V-iFluor™ 594None590/610 nm***100 Tests22826
Annexin V-iFluor™ 647None650/668 nm***100 Tests22827
Annexin V-mFluor™ Violet 450PI405/450 nm534/617 nm100 Tests22828
Annexin V-mFluor™ Violet 500PI414/508 nm534/617 nm100 Tests22829
Annexin V-mFluor™ Violet 550PI424/560 nm534/617 nm100 Tests22830
Annexin V-APCPI651/662 nm534/617 nm100 Tests22837
Annexin V-PENuclear Red™ DCS1565/575 nm631/651 nm100 Tests22838
Annexin V-FITCPI490/520 nm534/617 nm100 Tests22839
  1. For Cat No. 22825, 22826 and 22827 use Nuclear Blue™ DCS1 as a dead cell stain.

 

Product Ordering Information


Ordering information for recombinant annexin V, annexin V conjugates, and annexin V assay kits.

Cat No. Product Name Unit Size
20014 Annexin V, Recombinant 100 ug
20015 Annexin V, Recombinant 1 mg
20018 Annexin V-Biotin conjugate 100 tests
20030 Annexin V, FITC Labeled 100 tests
20031 Annexin V, TRITC Labeled 100 tests
20065 Annexin V-Cy3 conjugate 100 tests
20066 Annexin V-Cy5 conjugate 100 tests
20067 Annexin V-Cy5.5 conjugate 100 tests
20068 Annexin V-Cy7 conjugate 100 tests
20069 Annexin V-iFluor™ 633 conjugate 100 tests
20070 Annexin V-iFluor™ 350 conjugate 100 tests
20071 Annexin V-iFluor™ 488 conjugate 100 tests
20072 Annexin V-iFluor™ 555 conjugate 100 tests
20073 Annexin V-iFluor™ 594 conjugate 100 tests
20074 Annexin V-iFluor™ 647 conjugate 100 tests
20075 Annexin V-iFluor™ 680 conjugate 100 tests
20076 Annexin V-iFluor™ 750 conjugate 100 tests
20077 Annexin V-iFluor™ 700 conjugate 100 tests
20080 Annexin V-mFluor™ Violet 450 conjugate 100 tests
20081 Annexin V-mFluor™ Violet 510 conjugate 100 tests
20082 Annexin V-mFluor™ Violet 540 conjugate 100 tests
20085 Annexin V-mFluor™ Blue 570 conjugate 100 tests
20089 Annexin V, PacBlue conjugate 100 tests
20090 Annexin V, AF350 conjugate 100 tests
20092 Annexin V, AF488 conjugate 100 tests
20096 Annexin V, AF594 conjugate 100 tests
22824 Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry* 100 tests
22825 Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Orange Fluorescence Optimized for Flow Cytometry* 100 tests
22826 Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Red Fluorescence Optimized for Flow Cytometry* 100 tests
22827 Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Deep Red Fluorescence Optimized for Flow Cytometry* 100 tests
22828 Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Blue Fluorescence Excited at 405 nm* 100 tests
22829 Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Green Fluorescence Excited at 405 nm* 100 tests
22830 Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Orange Fluorescence Excited at 405 nm* 100 tests
22837 Cell Meter™ APC-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry* 100 tests
22838 Cell Meter™ PE-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry* 100 tests
22839 Cell Meter™ FITC-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry* 100 tests