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AAT Bioquest

Annexin-V Staining

Annexin V-FITC (Cat No. 20030) was used to identify levels of apoptosis in HeLa cells. HeLa cells were treated with low concentrations of EtOH to induce apoptosis and then stained with annexin V-FITC. The green label on the plasma membrane (Annexin V-FITC) indicates cells undergoing apoptosis.
Annexin V-FITC was used to identify levels of apoptosis in HeLa cells. HeLa cells were treated with low concentrations of EtOH to induce apoptosis and then stained with annexin V-FITC. The green label on the plasma membrane (Annexin V-FITC) indicates cells undergoing apoptosis.
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 specifically 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


HRP
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 (red). The nuclei of dead cells were labeled using Nuclear Green™ DCS1 (green). 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
XFD488 (Alexa Fluor® 488 equivalent)499520FITC730000.92100 tests (200 µL)20092
XFD594 (Alexa Fluor® 594 equivalent)590618Texas Red920000.66100 tests (200 µL)20096
FITC491516FITC730000.79100 tests (200 µL)20030
TRITC544570TRITC1000000.1100 tests (200 µL)20031
Cy3555569Cy3/TRITC1500000.04100 tests (200 µL)20065
Cy5656670Cy52500000.25100 tests (200 µL)20066
Cy5.5684701Cy5.52200000.23100 tests (200 µL)20067
Cy7756779Cy72500000.3100 tests (200 µL)20068
  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


Annexin V mFluor Violet 450
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.

Cell Meter Annexin V Binding Apoptosis Assay
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


Table 5. Ordering information for recombinant annexin V, annexin V conjugates, and annexin V assay kits.

Cat No.
Product Name
Unit Size
20014Annexin V, Recombinant100 ug
20015Annexin V, Recombinant1 mg
20018Annexin V-Biotin conjugate100 tests
20030Annexin V, FITC Labeled100 tests
20031Annexin V, TRITC Labeled100 tests
20065Annexin V-Cy3 conjugate100 tests
20066Annexin V-Cy5 conjugate100 tests
20067Annexin V-Cy5.5 conjugate100 tests
20068Annexin V-Cy7 conjugate100 tests
20069Annexin V-iFluor® 633 conjugate100 tests
20070Annexin V-iFluor® 350 conjugate100 tests
20071Annexin V-iFluor® 488 conjugate100 tests
20072Annexin V-iFluor® 555 conjugate100 tests
20073Annexin V-iFluor® 594 conjugate100 tests
20074Annexin V-iFluor® 647 conjugate100 tests
20075Annexin V-iFluor® 680 conjugate100 tests
20076Annexin V-iFluor® 750 conjugate100 tests
20077Annexin V-iFluor® 700 conjugate100 tests
20080Annexin V-mFluor™ Violet 450 conjugate100 tests
20081Annexin V-mFluor™ Violet 510 conjugate100 tests
20082Annexin V-mFluor™ Violet 540 conjugate100 tests
20085Annexin V-mFluor™ Blue 570 conjugate100 tests
20089Annexin V, PacBlue conjugate100 tests
20090Annexin V, AF350 conjugate100 tests
20092Annexin V, AF488 conjugate100 tests
20096Annexin V, AF594 conjugate100 tests
22824Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Green Fluorescence Optimized for Flow Cytometry*100 tests
22825Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Orange Fluorescence Optimized for Flow Cytometry*100 tests
22826Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Red Fluorescence Optimized for Flow Cytometry*100 tests
22827Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Deep Red Fluorescence Optimized for Flow Cytometry*100 tests
22828Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Blue Fluorescence Excited at 405 nm*100 tests
22829Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Green Fluorescence Excited at 405 nm*100 tests
22830Cell Meter™ Annexin V Binding Apoptosis Assay Kit *Orange Fluorescence Excited at 405 nm*100 tests
22837Cell Meter™ APC-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry*100 tests
22838Cell Meter™ PE-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry*100 tests
22839Cell Meter™ FITC-Annexin V Binding Apoptosis Assay Kit *Optimized for Flow Cytometry*100 tests
22870Annexin Binding Buffer *10X Optimized for Imaging and Flow Cytometry* 10 mL