Shop All Apoptosis Kits

ApoDETECT Annexin V-FITC Kit (Invitrogen™)

The ApoDETECT Annexin V-FITC Kit provides all the reagents needed to detect the apoptotic cells. Annexin V is a Ca2+-dependent phospholipid binding protein. This protein can bind to a variety of phospholipids, but it has the highest affinity for phosphatidylserine (PS). The ApoDETECT Annexin V-FITC Kit binds to negatively charged phospholipid surfaces in a Ca2+-dependent manner. It also prevents the formation of the prothrombinase complex, thereby inhibiting formation of thrombin (anticoagulant activity). The ApoDETECT Annexin V-FITC Kit can be used to detect apoptotic cells by flow cytometry or immunofluorescent cytology.

Kit Attributes:

Applications: Validated application for ApoDETECT Annexin V-FITC Kit is detecting apoptotic cells by flow cytometry or immunofluorescent cytology.
Format: Vial(s)
Detection Method: Fluorescence microscopy
Product Size: One kit

In normal cells phosphatidylserine (PS) is located on the inner leaflet of the plasma membrane. During the early stages of apoptosis, PS is translocated to the outer layer and is exposed on the external surface of the cell. This early event in apoptosis can be detected by using a sensitive method in which to detect PS exposure. Translocation of PS to the external surface of the plasma membrane is not a unique property of apoptotic cells, as this phenomenon also occurs during cell necrosis. However, during apoptosis, the cell membrane remains intact; whereas, during necrosis, the cell becomes leaky and loses its integrity. Therefore, it is necessary to assess membrane integrity together with PS translocation.

Annexin V is a Ca2+-dependent phospholipid binding protein. This protein can bind to a variety of phospholipids, but it has the highest affinity for phosphatidylserine (PS). Based on its affinity for PS, Annexin V can be utilized as a sensitive probe for cell surface exposure of PS. To use the Annexin V protein as a probe for apoptotic cells, the protein has been labeled with fluorescein isothiocyanate (FITC). In this form, the protein can be used directly for quantification of apoptotic cells. The measurement of Annexin V binding when performed simultaneously with a dye exclusion test (such as propidium iodide) can be used to effectively discriminate between apoptotic and necrotic cells.

Hypoxia Green Reagent for Flow Cytometry

Hypoxia Green Reagent for Flow Cytometry is an antibody-independent reagent used to detect low levels of oxygen levels in live cells. The membrane-permeant probe releases rhodamine as oxygen levels decrease, resulting in a fluorogenic response. Hypoxia is a characteristic of many diseases, including cardiovascular disease and tumor-mediated immunosuppression and is critical to tumor survival and growth.

• Sensitive, fixed, end-point assay
• Detects decreases in oxygen in live cells
• Fluorogenic, non-antibody based probe

CellEvent™ Caspase-3/7 Green Detection Reagent (Invitrogen™)

CellEvent™ Caspase-3/7 Green Detection Reagent is a novel fluorogenic substrate for activated caspase-3/7 that is compatible with both live cell and fixed imaging, with absorption/emission maxima of ~502/530 nm. Activation of caspase-3 is an essential event during apoptosis, making this an optimized reagent for analysis of apoptotic cells.

See more tools for detection of apoptotic cells >

• Optimized caspase-3/7 substrate for apoptosis analysis
• Easy to use—add and read; no cell lysis required
• Use for time course measurements; easily select the time point of interest
• Compatible with both live cell fluorescence-imaging and formaldehyde-based fixation methods
• Multiplex enabled—combine with other fluorescent reagents to confirm apoptosis in the same cell or cell population

Substrate specifics
CellEvent Caspase-3/7 Green Detection Reagent is a four-amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye with absorption/emission maxima of ~502/530 nm. The DEVD peptide sequence is a cleavage site for caspase-3/7, and the conjugated dye is non-fluorescent until cleaved from the peptide and bound to DNA.

Principles of action
CellEvent Caspase-3/7 Green Detection Reagent is intrinsically non-fluorescent as the DEVD peptide inhibits the ability of the dye to bind to DNA. However, after activation of caspase-3/7 in apoptotic cells, the DEVD peptide is cleaved, enabling the dye to bind to DNA and produce a bright, fluorogenic response. The fluorescent emission of the dye when bound to DNA is ~530 nm and can be observed using a standard FITC filter set.

Simple three-step protocol
To use CellEvent Caspase 3/7 Detection Reagent, simply add it to cells, incubate 30 minutes, and visualize (see figure). Apoptotic cells with activated caspase-3/7 will have bright green nuclei, while cells without activated caspase 3/7 will have minimal fluorescent signal (see figure).

Assay versatility allows for live cell detection and fixation
This robust assay enables the examination of caspase-3/7 activation in live cells. Furthermore, since wash steps are not necessary for detection, fragile apoptotic cells typically lost during wash steps are preserved. Importantly, the fluorescent signal from CellEvent Caspase 3/7 Detection Reagent can survive fixation and permeabilization. This allows for the flexibility to perform end-point assays and probe for other proteins of interest using immunocytochemistry.

Vybrant™ DyeCycle™ Violet/SYTOX™ AADvanced™ Apoptosis Kit, for flow cytometry (Invitrogen™)

The Vybrant™ DyeCycle™ Violet/SYTOX™ AADvanced™ apoptosis kit is used for the identification of apoptotic cells. This kit provides a rapid and convenient assay based upon fluorescence analysis of the compacted state of the nuclear chromatin in apoptotic cells. The kit contains solutions of the cell-permeant Vybrant™ DyeCycle™ Violet stain (Ex/Em max ~370/440nm) which is excited with UV or violet laser and SYTOX™ AADvanced™ (Ex/Em max ~546/650 nm) which is excited with a 488 nm laser. SYTOX™ AADvanced™ is used to detect changes in permeability of the plasma membrane therefore allowing identification of dead cells. The staining pattern resulting from simultaneous use of both dyes in combination make it possible to distinguish normal, apoptotic, and dead cell populations by flow cytometry.

View a selection guide for all apoptosis assays for flow cytometry.

Tali™ Apoptosis Kit - Annexin V Alexa Fluor™ 488 & Propidium Iodide (Invitrogen™)

The Tali® Apoptosis Kit enables identification of apoptotic cells and discrimination of apoptotic from necrotic and live cells in a population. The kit stains apoptotic cells with green Annexin V – Alexa Fluor® 488, stains necrotic cells with both red propidium iodide and green Annexin V – Alexa Fluor® 488, and does not stain live cells.

The Tali®Apoptosis Kit contains:

Annexin V – Alexa Fluor® 488 conjugate

• Annexin Binding solution
• Propidium Iodide (PI) solution

Apoptosis is a carefully regulated process of cell death that occurs as a normal part of development. Inappropriately regulated apoptosis is implicated in disease states, such as Alzheimer’s disease and cancer. Apoptosis is distinguished from necrosis by characteristic morphological and biochemical changes, including compaction and fragmentation of the nuclear chromatin, shrinkage of the cytoplasm, and loss of membrane asymmetry.

Easily Discriminate Apoptotic from Necrotic Cells with Two-color Staining
In normal live cells, phosphatidyl serine (PS) is located on the cytoplasmic surface of the cell membrane. In apoptotic cells, however, PS is translocated from the inner to the outer surface of the plasma membrane, exposing it to the extracellular environment. The human anticoagulant Annexin V displays a high affinity for PS. Annexin V labeled with a fluorophore such as Alexa Fluor® 488 can be used to identify apoptotic cells by binding to the exposed PS.

The Tali® Apoptosis Kit includes a solution of propidium Iodide (PI). PI is a cell-impermeant fluorogenic DNA-binding dye used for years to identify necrotic cells. PI is impermeant to live cells, but easily enters dead cells where it binds to nucleic acids and becomes fluorescent.

After staining a cell population using the Tali® Apoptosis Assay Kit with Annexin V-Alexa Fluor® 488 and Propidium Iodide, apoptotic cells display green fluorescence, dead cells display red and green fluorescence and live cells show little or no fluorescence.

eBioscience™ Annexin V Apoptosis Detection Kit eFluor™ 450 (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.

In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, or Fixable Viability Dyes such as eFluor™ 660 or eFluor™ 780. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).

Reported Application
Flow Cytometric Analysis

Image-iT™ LIVE Red Poly Caspases Detection Kit, for microscopy (Invitrogen™)

The Image-iT™ LIVE Red Poly Caspases Detection Kit employs a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA™) methodology, essentially an affinity label. The reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amine acid sequence. For poly caspases, this recognition sequence is valine-alanine-aspartic acid (VAD). A sulforhodamine group (SR) is attached as a reporter. The FLICA reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The FLICA inhibitor is cell permeant and noncytotoxic. Unbound FLICA molecules diffuse out of the cell and are washed away; the remaining red-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added.

eBioscience™ Annexin V Apoptosis Detection Kit PE (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.

In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, or Fixable Viability Dyes such as eFluor™ 660 or eFluor™ 780. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).

Note: Fixable Viability Dye eFluor™ 450 is not recommended for use with Annexin V Apoptosis Detection Kits.

Reported Application
Flow Cytometric Analysis

Pacific Blue™ Annexin V/SYTOX™ AADvanced™ Apoptosis Kit, for flow cytometry (Invitrogen™)

This product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to violet-fluorescent Pacific Blue™ dye and dead cells using SYTOX™ AADvanced™ stain. After staining a cell population with Pacific Blue™, annexin V and SYTOX™ AADvanced™, apoptotic cells show violet fluorescence, dead cells show red fluorescence, and live cells show little or no fluorescence. These populations are easily distinguished by a flow cytometer with the 405 nm and 488 nm lines for excitation. There is very little spectral overlap between the two dyes, therefore very little compensation is needed. Each kit contains sufficient reagents for approximately 50 flow cytometry tests.

View a selection guide for all apoptosis assays for flow cytometry.

CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit (Invitrogen™)

The CaspGLOW™ Fluorescein Active Caspase-3 Staining Kit contains the reagents to sensitively detect active Caspase-3 in mammalian cells. This assay utilizes the inhibitor specific for Caspase-3, DEVD-FMK, that is directly conjugated to FITC (fluorescein isothiocyanate) for the detection system. This reagent is cell permeable, non-toxic and irreversibly binds to the active enzyme. Detection of the labeled cells can be determined by flow cytometry, fluorescent microscopy or a fluorescent plate reader.

The Caspases are a family of aspartate-specific cysteine proteases that act in a step-wise signaling manner like kinases. Recruitment of these proteases to oligomerized receptors leads to activation and autoproteolytic cleavage. Active caspases can proteolyze additional caspases generating a caspase cascade. The final outcome of this signaling pathway is a form of controlled cell death, termed apoptosis.

Caspase-3, also known as Yama, CPP32, and apopain, cleaves its substrates at the carboxyl terminus of aspartate residues. Active Caspase-3 consists of 2 sets of homodimers (~ 17 and 12 kDa) that are derived from two precursor Caspase-3 polypeptides and has two active sites. Caspase-3 is proteolytically activated by other caspases.

Both subunits contribute to substrate binding and catalysis. The active site cysteine that covalently binds the substrate is located near the C-terminus of the large subunit. Active Caspase-3 has two-fold symmetry, two active site pockets each residing on an opposite side. Caspase-3, together with Caspases 8 and 9, is situated at pivotal junctions in apoptotic pathways. Caspase-3 appears to amplify Caspase 8 and 9 initiation signals into complete commitment to apoptotic disassembly.

Reported Application
Flow Cytometric Analysis, Immunocytochemistry

eBioscience™ Annexin V-FITC Apoptosis Detection Kit (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins, which bind to phosphatidylserine (PS) to identify apoptotic cells. In healthy cells, PS is predominantly located along the cytosolic side of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution in the phospholipid bilayer and translocates to the extracellular membrane, which is detectable with fluorescently labeled Annexin V. In early stages of apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI) and 7-AAD, therefore cells which display only Annexin V staining (PI/7-AAD negative) are in early stages of apoptosis. During late-stage apoptosis, loss of cell membrane integrity allows Annexin V binding to cytosolic PS, as well as cell uptake of PI and 7-AAD. Annexin V staining, paired with 7-AAD or PI is widely used to identify apoptotic stages by flow cytometry.

Host
E.coli

Conjugate
FITC

Laser
Blue Laser

Emit
520 nm

Excite
488 nm

Purity
> 98% pure

Molecular Mass
35.8 kDa

Reported Application
Flow Cytometric Analysis

eBioscience™ Annexin V Apoptosis Detection Kit FITC (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.

In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, or Fixable Viability Dyes such as eFluor™ 660 or eFluor™ 780. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).

Note: Fixable Viability Dye eFluor™ 450 is not recommended for use with Annexin V Apoptosis Detection Kits.

Reported Application
Flow Cytometric Analysis

eBioscience™ Annexin V Apoptosis Detection Kit PerCP-eFluor™ 710 (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS) in all mammalian species. Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.

In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, or Fixable Viability Dyes such as eFluor™ 660 or eFluor™ 780. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).

Due to the emission spectrum of PerCP-eFluor™ 710, the Annexin V Apoptosis Detection Kit PerCP-eFluor™ 710 is not compatible with propidium iodide and 7-AAD. It is recommended to substitute a Fixable Viability Dye such as eFluor™ 660 or eFluor™ 780 in their place.

Not included:
Fixable Viability Dye eFluor™ 660 (cat. 65-0864)
Fixable Viability Dye eFluor™ 780 (cat. 65-0865)

Note: Fixable Viability Dye eFluor™ 450 is not recommended for use with Annexin V Apoptosis Detection Kits.

Reported Application
Flow Cytometric Analysis

eBioscience™ Annexin V-Biotin Apoptosis Detection Kit (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins, which bind to phosphatidylserine (PS) to identify apoptotic cells. In healthy cells, PS is predominantly located along the cytosolic side of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution in the phospholipid bilayer and translocates to the extracellular membrane, which is detectable with fluorescently labeled Annexin V. In early stages of apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI) and 7-AAD, therefore cells which display only Annexin V staining (PI/7-AAD negative) are in early stages of apoptosis. During late-stage apoptosis, loss of cell membrane integrity allows Annexin V binding to cytosolic PS, as well as cell uptake of PI and 7-AAD. Annexin V staining, paired with 7-AAD or PI is widely used to identify apoptotic stages by flow cytometry.

Host
E.coli

Conjugate
Biotin

Purity
> 98% pure

Molecular Mass
35.8 kDa

Reported Application
Flow Cytometric Analysis

eBioscience™ Annexin V Apoptosis Detection Kit APC (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.

In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, or Fixable Viability Dyes such as eFluor™ 660 or eFluor™ 780. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).

Note: Fixable Viability Dye eFluor™ 450 is not recommended for use with Annexin V Apoptosis Detection Kits.

Reported Application
Flow Cytometric Analysis

Apo-Direct Apoptosis Detection Kit (Invitrogen™)

The APO-DIRECT™ Kit is a 2-color staining method for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry. The kit contains the instructions and reagents required for measuring apoptosis in cells including positive and negative control cells for assessing reagent performance; washing, reaction and rinsing buffers for processing individual steps in the assay; terminal deoxynucleotidyl transferase enzyme (TdT), Fluorescein-deoxyuridine triphosphate and propidium iodide/RNase A solution for counterstaining the total DNA.

One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of DNA laddering when the DNA is analyzed by agarose gel electrophoresis. The DNA of non-apoptotic cells that remains largely intact does not display this laddering on agarose gels during electrophoresis. The large number of DNA fragments appearing in apoptotic cells results in a multitude of 3'-hydroxyl termini in the DNA. This property can be used to identify apoptotic cells by labeling the 3'-hydroxyl ends with directly conjugated fluorescein- deoxyuridine triphosphate nucleotides (FITC-dUTP). The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes a template-independent addition of deoxyribonucleoside triphosphates to the 3'-hydroxyl ends of double- or single-stranded DNA with either blunt, recessed or overhanging ends. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized in the APO-DIRECT™ Kit. Non-apoptotic cells do not incorporate significant amounts of the FITC-dUTP due to the lack of exposed 3'-hydroxyl DNA ends.

Sufficientreagents are provided to process 50 cell suspensions including 5 mL positive and 5 mL negative control cell suspensions of approximately 1x106 cells per mL in 70% (v/v) ethanol.

Reported Application
Immunocytochemistry, Flow Cytometric Analysis

Vybrant™ Apoptosis Assay Kit #6, Biotin-X Annexin V/Alexa Fluor™ 350 Streptavidin/Propidium Iodide (Invitrogen™)

The Vybrant Apoptosis Assay Kit #6 uses the bright, blue-fluorescent Alexa Fluor 350 conjugate of streptavidin in conjunction with biotin-X annexin V to detect apoptotic cell populations either by flow cytometry or imaging. Alexa Fluor 350 streptavidin binds to biotin-X annexin V, which in turn binds to the phosphatidylserine on the surfaces of apoptotic cells. Necrotic cells are distinguished using red-fluorescent propidium iodide, which binds tightly to the nucleic acids in those cells. Propidium iodide does not penetrate the plasma membranes of live cells or early apoptotic cells. The kit contains an ample amount of each reagent for about 50 flow cytometric assays.

View a selection guide for all apoptosis assays for flow cytometry.

Image-iT™ LIVE Red Caspase-3 and -7 Detection Kit, for microscopy (Invitrogen™)

The Image-iT™ LIVE Red Caspase-3 and -7 Detection Kit employs a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA™) methodology, essentially an affinity label. The reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amine acid sequence. For caspase-3 and -7, this recognition sequence is aspartic acid-glutamic acid-valine-aspartic acid (DEVD). A sulforhodamine group (SR) is attached as a reporter. The FLICA reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The FLICA inhibitor is cell permeant and noncytotoxic. Unbound FLICA molecules diffuse out of the cell and are washed away; the remaining red-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added.

Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 647 dye (Invitrogen™)

The Click-iT® Plus TUNEL assay detects apoptotic cells in tissue and cultured cell samples through the use of a small, highly specific labeling moiety and a bright fluorescent dye. After incorporation of the labeling moiety into DNA fragments, detection is achieved through a catalyzed “click” reaction using conditions mild enough to preserve the fluorescent signal from GFP or RFP.

More tools for apoptosis detection and measurement >

• Optimized for the detection of apoptotic cells in either tissue or cell samples
• Multiplex enabled—optimized to work with fluorescent dyes or proteins such as GFP and RFP
• Improved TUNEL assay—better label incorporation due to small reactive moiety
• Bright apoptotic signal—uses Alexa Fluor® dyes, resulting in a stable, non-photobleaching fluorescent signal
• Flexibility—the assay can be configured to test from 1 to 50 samples at a time

Fragmentation of cellular DNA is a hallmark of apoptosis. The TUNEL assay is the most widely used method to detect fragmented DNA in apoptotic cells in tissue samples. The TUNEL assay begins with incorporation of modified dUTP at the 3’-OH end of the fragmented DNA. The dUTP modification is often the addition of a fluorophore. And due to the size of the fluorophore, the modified dUTP can display lower than expected incorporation rates. These lower incorporation rates can affect the sensitivity of the TUNEL assay. Additionally, the fluorophores used in currently available TUNEL assays suffer from photobleaching and fluorescent spectral overlap issues, both of which reduce sensitivity and ability to multiplex the assay.

The Click-iT® Plus TUNEL assay was developed to address these issues. The assay uses dUTP modified with an alkyne group (a small bio-orthogonal functional group) that allows the nucleotide to be more readily incorporated. After incorporation, a highly specific click reaction between the alkyne group and an Alexa Fluor® picolyl azide fluorescent dye, and subsequent detection of that dye, results in a sensitive and specific assay for the detection of apoptotic cells. Because of the gentle reaction conditions, the Click-iT® Plus TUNEL assay enables multiplexing with fluorescent proteins or dyes.

The Click-iT® Plus TUNEL assay has been validated with several different formalin-fixed paraffin-embedded tissue types. In all cases the ability to multiplex with fluorescent proteins and dyes was preserved. Additionally, the ability to stain actin using fluorescent-labeled phalloidin was also preserved.

The Click-iT® Plus TUNEL assay contains all of the reagents needed to detect apoptotic cells from either tissue or cell samples. The reagents supplied in this kit can be used to test 50 samples and can be configured to test from 1 to 50 samples at a time.

Vybrant™ Apoptosis Assay Kit #5, Hoechst 33342/Propidium Iodide (Invitrogen™)

The Vybrant Apoptosis Assay Kit #5 provides a rapid and convenient assay for apoptosis based on fluorescence detection of the compacted state of the chromatin in apoptotic cells. This kit contains ready-to-use solutions of blue-fluorescent Hoechst 33342 dye, which stains the condensed chromatin of apoptotic cells more brightly than the chromatin of nonapoptotic cells, and red-fluorescent propidium iodide dye, which stains dead cells.

CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit (Invitrogen™)

The CellEvent™ Caspase-3/7 Green Flow Cytometry Assay Kit enables the flow cytometric detection of activated caspase-3 and caspase-7 in apoptotic cells. The kit includes the novel fluorogenic substrate CellEvent™ Caspase-3/7 Green Detection Reagent, as well as SYTOX™ AADvanced™ Dead Cell Stain.

View a selection guide for all apoptosis assays for flow cytometry >

• Caspase-3/7 specific—includes the recognition sequence for activated caspase-3 and caspase-7
• Easy identification—clearly identify live, dead, and apoptotic cell populations
• Quick analysis—washing and fixation is not required
• Multicolor compatibility—combine with other dyes excitable by the 488 nm laser or other lasers

CellEvent Caspase-3/7 Green Detection Reagent is a cell-permeant reagent that consists of a four-amino acid peptide (DEVD, a caspase 3/7 recognition sequence) conjugated to a nucleic acid-binding dye. During apoptosis, caspase-3 and caspase-7 proteins are activated and cleave the peptide. Cleavage of the peptide allows binding of DNA by the reagent, thereby labeling the apoptotic cells with a bright, fluorogenic signal that has absorption/emission maxima of ~502/530 nm. When used together with the SYTOX AADvanced Dead Cell Stain, apoptotic cells can be easily discriminated from live and necrotic cells.

Because no single parameter defines apoptosis in all systems, we strongly suggest using a combination of different measurements for reliable detection of apoptosis. For more information, see Apoptosis Reagents in Flow Cytometry.

Click-iT™ TUNEL Alexa Fluor™ 488 Imaging Assay, for microscopy & HCS (Invitrogen™)

The Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay utilizes a dUTP modified with an alkyne, a small, bio-orthogonal functional group that enables the nucleotide to be more readily incorporated by TdT than other modified nucleotides, including BrdUTP, biotin-dUTP or fluorescein-dUTP. Detection is based on a click reaction, a copper (I) catalyzed reaction between an azide and alkyne. Click chemistry fills the void when methods such as direct labeling or the use of antibodies are not efficient. The small size of the detection reagent, an Alexa Fluor® azide (MW <~1000) compared to that of an antibody (MW ~150,000) enables facile penetration of complex samples with only mild fixation and permeabilization required.

Compared to other assays using one of the other modified nucleotides, the Click-iT® TUNEL assay is able to detect a higher percentage of apoptotic cells under identical conditions. The assay is also fast and is complete within 2 hours. Furthermore, the Click-iT® TUNEL assay allows multiplexing with surface and intracellular biomarker detection.

More tools for apoptosis detection and measurement >

Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit, for flow cytometry (Invitrogen™)

The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit provides a simple and fast method for the detection of apoptosis with dead cell discrimination by flow cytometry. The Violet Ratiometric Membrane Asymmetry Probe, 4'-N,N-diethylamino-6-(N,N,N-dodecyl-methylamino-sulfopropyl)-methyl-3-hydroxyflavone (F2N12S), is a novel violet excitable dye for the detection of membrane asymmetry changes during apoptosis. The dye exhibits an excited-state intramolecular proton transfer (ESIPT) reaction resulting in a dual fluorescence with two emission bands corresponding to 530 nm and 585 nm, producing a two-color ratiometric response to variations in surface charge. Ratiometric probes have several advantages over traditional fluorochrome labeled reagents. The ratiometric probe is a self-calibrating absolute parameter of apoptotic transformation, which is independent of probe concentration, cell size, and instrument variation, such as fluctuations of laser intensity or sensitivity of the detectors. Given that apoptosis modifies the surface charge of the outer leaflet of the plasma membrane the violet membrane asymmetry probe F2N12S can monitor changes in membrane asymmetry that occur during apoptosis through a change in the relative intensity of the two emission bands of the dye. The F2N12S probe is combined with SYTOX(R) AADvanced dead cell stain, which is capable of passing through the cell membrane only in late apoptotic or necrotic cells allowing discrimination form early apoptotic cells. Samples can be analyzed after a 5 minute incubation at room temperature and does not require special buffers or wash steps. This kit can be paired with other reagents such as MitoProbe™ DiIC1(5) or annexin V for multiparametric analysis of apoptosis and viability. This reagent kit allow researchers to maximaize the utility of their instruments by utilizing the violet laser Each kit contains sufficient reagents for ~100 flow cytometry tests.

View a selection guide for all apoptosis assays for flow cytometry.

Image-iT™ LIVE Green Poly Caspases Detection Kit, for microscopy (Invitrogen™)

The Image-iT™ LIVE Green Poly Caspases Detection Kit employs a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA™) methodology, essentially an affinity label. The reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amine acid sequence. For poly caspases, this recognition sequence is valine-alanine-aspartic acid (VAD). A fluorescein group is attached as a reporter. The FLICA reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The FLICA inhibitor is cell permeant and noncytotoxic. Unbound FLICA molecules diffuse out of the cell and are washed away; the remaining green-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added.

CaspGLOW™ Fluorescein Active Caspase-8 Staining Kit (Invitrogen™)

The CaspGLOW™ Fluorescein Active Caspase-8 Staining Kit contains all the reagents necessary to detect active caspase-8 in cells with high sensitivity. Fluorescein (FITC)-conjugated IETD-FMK, a specific inhibitor of caspase-8, is utilized in this assay for detection. This reagent is cell permeable, non-toxic and irreversibly binds to the active enzyme. Detection of the labeled cells can be determined by flow cytometry, fluorescent microscopy or a fluorescent plate reader.

The caspases constitute a family of aspartate-specific cysteine proteases that mediate a sequence of cleavage events. Recruitment of the inactive proenzyme to oligomerized receptors leads to caspase activation and autoproteolytic cleavage. These active enzymes can then cleave other caspases, thereby generating a caspase signaling cascade that leads to a form of programmed cell death termed apoptosis.

Caspase-8, which is also known as FLICE, MACHalpha1, and Mch5, cleaves its substrates at the C-terminal aspartic acid residue of the motif Asp-X-X-Asp. Eight isoforms of caspase-8 exist, of which caspase-8/a and 8/b are the predominant forms. Upon stimulation of death receptors such as CD95/APO-1/Fas, TRAIL-R1, TRAIL-R2, TNFR1, and TRAMP, caspase-8 is recruited to the death-inducing signaling complex (DISC). Subsequent dimerization leads to caspase-8 activation via autocatalytic cleavage, which leads to the formation of a 12-kDa prodomain and a 43-kDa intermediate fragment that is further cleaved to produce 26-kDa and 18-kDa active enzymes. As an initiator caspase, this enzyme initiates a caspase signaling cascade that results in apoptosis. Substrates of caspase-8 include caspases-3 and -7, as well as the pro-apoptotic Bcl-2 family member Bid. In addition to its role in cell death, caspase-8 has been linked to cell adhesion and motility.

Reported Application
Flow Cytometric Analysis, Immunocytochemistry

MitoProbe™ TMRM Assay Kit for Flow Cytometry

The MitoProbe TMRM Assay Kit contains tetramethylrhodamine methyl ester (TMRM) for the detection of mitochondrial membrane potential state and CCCP for the induction of mitochondrial membrane depolarization, if desired.

• Study mitochondrial membrane potential in live cells
• Quick, easy protocol

Determining the state of mitochondrial health can be achieved by detecting changes in the mitochondrial membrane potential. In a healthy cell with active mitochondria, TMRM is readily sequestered, thereby emitting a red-orange fluorescent signal. When apoptosis is induced or a treatment like CCCP is applied, the mitochondrial membrane is depolarized and the TMRM signal diminishes.

Vybrant™ FAM Caspase-8 Assay Kit, for flow cytometry (Invitrogen™)

The Vybrant™ FAM Caspase-8 Assay Kit employs a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA™) methodology, essentially an affinity label. The reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amine acid sequence. For caspase-8, this recognition sequence is leucine-glutamic acid-threonine-aspartic acid (LETD). A fluorescein group is attached as a reporter. The FLICA reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The FLICA inhibitor is cell permeant and noncytotoxic. Unbound FLICA molecules diffuse out of the cell and are washed away; the remaining green-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added. This kit includes the FLICA reagent specific for caspase-8, Hoechst 33342 stain, and propidium iodide stain, which allows the simultaneous evaluation of caspase activation, membrane permeability, and cell cycle by flow cytometry.

View a selection guide for all apoptosis assays for flow cytometry.

CellEvent™ Caspase-3/7 Green ReadyProbes™ Reagent (Invitrogen™)

CellEvent® Caspase-3/7 Green ReadyProbes® Reagent is a fluorogenic, no-wash indicator of activated caspase-3/7 for live- and fixed-cell applications. Activation of caspase-3 is an early indicator of apoptosis and CellEvent® Caspase-3/7 Green reagent allows rapid and sensitive detection of cells destined for cell death.

• Simple and fast no-wash protocol helps preserve delicate apoptotic cells
• Compatible with both live-cell fluorescence imaging and formaldehyde-based fixation methods
• Ready-to-use liquid formulation in convenient dropper bottle—no need to dilute, weigh, or pipette
• Stability at room temperature—keep handy at your scope or cell culture area.

See other ReadyProbes® reagents for cell staining
Learn more about other assays for apoptosis

Cell imaging applications
CellEvent® Caspase-3/7 Green reagent is a four amino acid peptide (DEVD) conjugated to a nucleic acid-binding dye that is non-fluorescent when not bound to DNA. The CellEvent® Caspase-3/7 Green reagent is intrinsically non-fluorescent, as the DEVD peptide inhibits binding of the dye to DNA. Upon activation of caspase-3/7 in apoptotic cells, the DEVD peptide is cleaved and the free dye can bind DNA, generating a bright green fluorescence. The fluorescence emission of the dye when bound to DNA is 530 nm and can be observed using a standard FITC filter set.

Suggestions for use
• In most cases, 2 drops/ml and an incubation time of 30 minutes is sufficient for bright nuclear staining of apoptotic cells; however, optimization may be needed for some cell types, conditions, and applications. In such cases, simply add more or fewer drops until the optimal staining intensity is obtained.
• If desired, combine CellEvent™ Caspase-3/7 Green ReadyProbes® reagent with a red or far-red nuclear stain for a total cell count
• CellEvent™ Caspase-3/7 Green dye is excited with a maximum at 502 nm and has an emission maximum at 530 nm. It is detected through standard GFP and FITC filters.

Dead Cell Apoptosis Kit with Annexin V PE and SYTOX™ Green, for flow cytometry (Invitrogen™)

This product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to the orange fluorescent phycobiliprotein R-PE, and dead cells using SYTOX™ Green nucleic acid stain. After treatment with both probes, apoptotic cells show orange fluorescence, dead cells show green fluorescence, and live cells show little or no fluorescence. These populations can easily be distinguished in the 530/30 nm and 585/42 nm bandpass filters with a 488 nm laser flow cytometer.

View a selection guide for all apoptosis assays for flow cytometry.

EnzChek™ Caspase-3 Assay Kit #2, Z-DEVD-R110 substrate (Invitrogen™)

The EnzChek® Caspase-3 Assay Kit #2 enables detection of apoptosis by providing a simple and reliable method for assaying caspase-3/7 activity. The kit can be used to continuously measure the activity of caspase-3/7 in cell extracts and purified enzyme preparations, using a fluorometer or fluorescence microplate reader.

See our complete line of Fluorescence Microplate assays.

• Fluorescent assay using standard fluorescein (FITC) excitation/emission settings
• Format allows for continuous measurement of caspase-3/7 activity in cell extracts
• Fluorescent and enzymatic controls included

The rhodamine 110-derived substrate (Z-DEVD-R110) used in this assay is a non-fluorescent bisamide compound that, upon enzymatic cleavage, is converted in a two-step process to the fluorescent monoamide and then to the even more fluorescent R110 product. Both of these hydrolysis products exhibit spectral properties similar to those of fluorescein, with peak excitation and emission wavelengths of 496 nm and 520 nm, respectively.

In addition to the Z-DEVD–R110 substrate, the EnzChek® Caspase Assay Kit #2 contains the reversible aldehyde inhibitor Ac-DEVD-CHO, as well as the reference standard R110. The Ac-DEVD-CHO inhibitor confirms that the fluorescence signals in both induced and control cell populations are due to the activity of caspase-3/7. The reference standard allows for quantification of the amount of R110 released in the reaction.

eBioscience™ Annexin V Apoptosis Detection Set PE-Cyanine7 (Invitrogen™)

Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS) in all mammalian species. Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.

In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD, or Fixable Viability Dyes such as eFluor™ 660 or eFluor™ 780. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).

Due to the emission spectrum of PE-Cyanine7, the Annexin V Apoptosis Detection Set PE-Cyanine7 is not compatible with propidium iodide and 7-AAD. It is recommended to substitute a Fixable Viability Dye such as eFluor™ 660 or eFluor™ 780 in their place.

Not included:
Fixable Viability Dye eFluor™ 660 (cat. 65-0864)
Fixable Viability Dye eFluor™ 780 (cat. 65-0865)

Note: Fixable Viability Dye eFluor™ 450 is not recommended for use with Annexin V Apoptosis Detection Kits.

Reactivity/Species
Human, Mouse, Rat

Reported Application
Flow Cytometric Analysis

CaspGLOW™ Fluorescein Active Caspase Staining Kit (Invitrogen™)

The CaspGLOW™ Fluorescein Active Staining Kit contains the reagents to sensitively detect active caspases in mammalian cells. This assay utilizes the inhibitor specific for all caspases, Z-VAD-FMK, that is directly conjugated to FITC (fluorescein isothiocyanate) for the detection system. This reagent is cell permeable, non-toxic and irreversibly binds to the active enzyme. Detection of the labeled cells can be determined by flow cytometry, fluorescent microscopy or a fluorescent plate reader.

The Caspases are a family of aspartate-specific cysteine proteases that act in a step-wise signaling manner like kinases. Recruitment of these proteases to oligomerized receptors leads to activation and autoproteolytic cleavage. Active caspases can proteolyze additional caspases generating a caspase cascade. The final outcome of this signaling pathway is a form of controlled cell death, termed apoptosis.

Reported Application
Flow Cytometric Analysis, Immunocytochemistry

Click-iT™ TUNEL Alexa Fluor™ 647 Imaging Assay, for microscopy & HCS (Invitrogen™)

The Click-iT® TUNEL Alexa Fluor® 647 Imaging Assay utilizes a dUTP modified with an alkyne, a small, bio-orthogonal functional group that enables the nucleotide to be more readily incorporated by TdT than other modified nucleotides, including BrdUTP, biotin-dUTP or fluorescein-dUTP. Detection is based on a click reaction, a copper (I) catalyzed reaction between an azide and alkyne. Click chemistry fills the void when methods such as direct labeling or the use of antibodies are not efficient. The small size of the detection reagent, an Alexa Fluor® azide (MW <~1000) compared to that of an antibody (MW ~150,000) enables facile penetration of complex samples with only mild fixation and permeabilization required.

Compared to other assays using one of the other modified nucleotides, the Click-iT® TUNEL assay is able to detect a higher percentage of apoptotic cells under identical conditions. The assay is also fast and is complete within 2 hours. Furthermore, the Click-iT® TUNEL assay allows multiplexing with surface and intracellular biomarker detection.

More tools for apoptosis detection and measurement >

Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 594 dye (Invitrogen™)

The Click-iT® Plus TUNEL assay detects apoptotic cells in tissue and cultured cell samples through the use of a small, highly specific labeling moiety and a bright fluorescent dye. After incorporation of the labeling moiety into DNA fragments, detection is achieved through a catalyzed “click” reaction using conditions mild enough to preserve the fluorescent signal from GFP or RFP.

More tools for apoptosis detection and measurement >

• Optimized for the detection of apoptotic cells in either tissue or cell samples
• Multiplex enabled—optimized to work with fluorescent dyes or proteins such as GFP and RFP
• Improved TUNEL assay—better label incorporation due to small reactive moiety
• Bright apoptotic signal—uses Alexa Fluor® dyes, resulting in a stable, non-photobleaching fluorescent signal
• Flexibility—the assay can be configured to test from 1 to 50 samples at a time

Fragmentation of cellular DNA is a hallmark of apoptosis. The TUNEL assay is the most widely used method to detect fragmented DNA in apoptotic cells in tissue samples. The TUNEL assay begins with incorporation of modified dUTP at the 3’-OH end of the fragmented DNA. The dUTP modification is often the addition of a fluorophore. And due to the size of the fluorophore, the modified dUTP can display lower than expected incorporation rates. These lower incorporation rates can affect the sensitivity of the TUNEL assay. Additionally, the fluorophores used in currently available TUNEL assays suffer from photobleaching and fluorescent spectral overlap issues, both of which reduce sensitivity and ability to multiplex the assay.

The Click-iT® Plus TUNEL assay was developed to address these issues. The assay uses dUTP modified with an alkyne group (a small bio-orthogonal functional group) that allows the nucleotide to be more readily incorporated. After incorporation, a highly specific click reaction between the alkyne group and an Alexa Fluor® picolyl azide fluorescent dye, and subsequent detection of that dye, results in a sensitive and specific assay for the detection of apoptotic cells. Because of the gentle reaction conditions, the Click-iT® Plus TUNEL assay enables multiplexing with fluorescent proteins or dyes.

The Click-iT® Plus TUNEL assay has been validated with several different formalin-fixed paraffin-embedded tissue types. In all cases the ability to multiplex with fluorescent proteins and dyes was preserved. Additionally, the ability to stain actin using fluorescent-labeled phalloidin was also preserved.

The Click-iT® Plus TUNEL assay contains all of the reagents needed to detect apoptotic cells from either tissue or cell samples. The reagents supplied in this kit can be used to test 50 samples and can be configured to test from 1 to 50 samples at a time.

Apo-BrdU Apoptosis Detection Kit (Invitrogen™)

The APO-BRDU™ Kit is a 2-color staining method for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry. The kit contains the instructions and reagents required for measuring apoptosis in cells, including positive and negative control cells for assessing reagent performance; washing, reaction and rinsing buffers for processing individual steps in the assay; terminal deoxynucleotidyl transferase enzyme (TdT), bromodeoxyuridine triphosphate (Br-dUTP), and fluorescein labeled anti-BrdU antibody for labeling DNA breaks and propidium iodide/RNase A solution for counterstaining the total DNA.

One of the most easily measured features of apoptotic cells is the break-up of the genomic DNA by cellular nucleases. These DNA fragments can be extracted from apoptotic cells and result in the appearance of DNA laddering when the DNA is analyzed by agarose gel electrophoresis. The DNA of non-apoptotic cells that remains largely intact does not display this laddering on agarose gels during electrophoresis. The large number of DNA fragments appearing in apoptotic cells results in a multitude of 3'-hydroxyl termini in the DNA. This property can be used to identify apoptotic cells by labeling the 3'-hydroxyl ends with bromolated deoxyuridine triphosphate nucleotides (Br-dUTP). The enzyme terminal deoxynucleotidyl transferase (TdT) catalyzes a template independent addition of deoxyribonucleoside triphosphates to the 3'-hydroxyl ends of double- or single-stranded DNA with either blunt, recessed or overhanging ends. A substantial number of these sites are available in apoptotic cells providing the basis for the method utilized in the APO-BRDU™ Kit. Recent evidence has demonstrated that Br-dUTP is more readily incorporated into the genome of apoptotic cells than are the deoxynucleotide triphosphates complexed to larger ligands like fluorescein, biotin or digoxigenin. This greater incorporation gives rise to a brighter flow cytometry signal when the Br-dUTP sites are identified by a fluorescein labeled anti-BrdU monoclonal antibody. Non-apoptotic cells do not incorporate significant amounts of the Br-dUTP due to the lack of exposed 3'-hydroxyl DNA ends.

Sufficient reagents are provided to process a total of 60 cell suspensions including 5 mL positive and 5 mL negative control cell suspensions of approximately 1x106 cells per mL in 70% (v/v) ethanol.

Reported Application
Immunocytochemistry, Flow Cytometric Analysis

Vybrant™ Apoptosis Assay Kit #4, YO-PRO™-1/Propidium Iodide (Invitrogen™)

The Vybrant Apoptosis Assay Kit #4 detects apoptosis on the basis of changes that occur in the permeability of cell membranes. This kit contains ready-to-use solutions of both YO-PRO-1 and propidium iodide nucleic acid stains. YO-PRO-1 stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained red-fluorescent with propidium iodide.

View a selection guide for all apoptosis assays for flow cytometry.

Chromatin Condensation & Membrane Permeability Dead Cell Apoptosis Kit with Hoechst 33342, YO-PRO™-1, and PI dyes, for flow cytometry (Invitrogen™)

This product detects apoptotic cells with changes in nuclear chromatin condensation and plasma membrane permeability, using three nucleic acid stains: UV-excitable Hoechst 33342, green fluorescent YO-PRO™ dye, and propidium iodide. The YO-PRO™ dye can enter apoptotic cells, whereas red-fluorescent propidium iodide (PI) cannot. Thus after staining with YO-PRO™-1 dye and PI, apoptotic cells show green fluorescence, and dead cells show primarily red fluorescence and some green fluorescence. Blue fluorescent Hoechst 33342 brightly stains the condensed chromatin of apoptotic cells and more dimly stains the normal chromatin of live cells. The staining pattern resulting from the simultaneous use of these three dyes makes it possible to distinguish normal, apoptotic and dead cell populations by flow cytometry or fluorescence microscopy.

View a selection guide for all apoptosis assays for flow cytometry.

Vybrant™ FAM Caspase-3 and -7 Assay Kit, for flow cytometry (Invitrogen™)

The Vybrant™ FAM Caspase-3 and -7 Assay Kit employs a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA™) methodology, essentially an affinity label. The reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amine acid sequence. For caspase-3 and -7, this recognition sequence is aspartic acid-glutamic acid-valine-aspartic acid (DEVD). A fluorescein group is attached as a reporter. The FLICA reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The FLICA inhibitor is cell permeant and noncytotoxic. Unbound FLICA molecules diffuse out of the cell and are washed away; the remaining green-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added. This kit includes the FLICA reagent specific for caspase-3 and -7, Hoechst 33342 stain, and propidium iodide stain, which allows the simultaneous evaluation of caspase activation, membrane permeability, and cell cycle by flow cytometry.

View a selection guide for all apoptosis assays for flow cytometry.

Click-iT™ TUNEL Colorimetric IHC Detection Kit (Invitrogen™)

The Click-iT™ TUNEL Colorimetric IHC Detection Kit is used to identify apoptotic cells in tissue and cultured cell samples through the use of a small, highly specific labeling moiety and streptavidin-peroxidase conjugation. After incorporation of the labeling moiety into sites of DNA fragmentation, detection is achieved through a catalyzed "click" reaction that adds a biotin group at these sites. The subsequent addition of a streptavidin-peroxidase and peroxidase substrate results in a dark brown signal that can be detected with light microscopy and stored for future analysis.

Find more tools for image-based detection of apoptotic cells >

• Optimized for the detection of apoptotic cells in either tissue or cell samples
• Improved colorimetric TUNEL assay—better label incorporation due to small reactive moiety
• Increased sensitivity—specific label incorporation results in lower background and brighter signal
• Content-rich results—cell morphology, cellular environment, and apoptotic signal are clearly visible
• Flexibility—the assay can be configured for 50 independent TUNEL apoptosis tests

The later stages of apoptosis are characterized by changes in nuclear morphology, chromatin condensation, nuclear envelope degradation, and DNA fragmentation. Terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assays are based on the incorporation of modified dUTPs by terminal deoxynucleotidyl transferase (TdT) at the 3’-OH ends of fragmented DNA. Colorimetric TUNEL assays utilize dUTPs conjugated with a biotin moiety, followed by the addition of a streptavidin-peroxidase conjugate and peroxidase substrate, resulting in a dark brown apoptotic signal. However, colorimetric TUNEL assays are prone to high background, which reduces the sensitivity and specificity of the signal.

The Click-iT TUNEL Colorimetric IHC Detection Kit was developed to address these issues by utilizing a dUTP modified with an alkyne group (a small bio-orthogonal functional group) that enables the nucleotide to be more readily incorporated by TdT. After incorporation, a highly specific click reaction covalently links biotin azide and the alkyne group. Streptavidin-peroxidase (horseradish peroxidase) followed by peroxidase substrate (DAB) are added, allowing colorimetric detection of apoptotic cells. The high degree of labeling specificity inherent in the click technology results in low background and improved detection of apoptotic cells.

The Click-iT TUNEL Colorimetric IHC Detection Kit has been optimized and contains all of the reagents needed for detection of apoptotic cells from either tissue or cell samples. The reagents supplied in this kit can be used to test 50 samples and configured to test from one to 50 samples at a time.

EnzChek™ Caspase-3 Assay Kit #1, Z-DEVD-AMC substrate (Invitrogen™)

The EnzChek Caspase-3 Assay Kit #1 allows detection of apoptosis by providing a simple and reliable method for assaying caspase-3 activity. The basis for the assay is the aminomethylcoumarin (AMC)-derived substrate Z-DEVD-AMC. This substrate, which is weakly fluorescent in the UV range (excitation/emission maxima ~330/390 nm), yields a bright, blue-fluorescent product (excitation/emission maxima ~342/441 nm) upon proteolytic cleavage. The kit can be used to continuously measure the activity of caspase-3 and closely related proteases in cell extracts and purified enzyme preparations, using a fluorometer or fluorescence microplate reader.

Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide (PI) (Invitrogen™)

The Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 & Propidium Iodide is a flow cytometry kit used to measure early apoptosis by detecting phosphatidyl serine expression and membrane permeability.

View a selection guide for all apoptosis assays for flow cytometry.

Superior brightness
Unlike other Annexin V kits that have lower protein concentrations or purification levels, the Annexin V Alexa Fluor™ 488 conjugate is optimized for flow cytometry to provide the largest separation between apoptotic and live cells. The Alexa Fluor™ 488 dye is a superior green-fluorescent dye with a spectrum similar to fluorescein (FITC).

High binding efficiency
Annexin V conjugates are made from a highly purified cys-annexin, which leads to higher binding efficiency, resulting in highly accurate characterization of the apoptotic process.

Multi-parametric
Many publications require a minimum of two different ways to identify that cells are apoptotic. This multi-parametric kit detects phosphatidyl serine (PS) on the cytoplasmic surface of the cell membrane and membrane integrity using propidium iodide.

How it works
When cells are stained with Annexin V and propidium iodide, apoptotic cells expressing PS show green fluorescence, which can be detected in the FITC channel, and low red fluorescence. Dead or necrotic cells show bright red fluorescence and no green fluorescence, while live cells show no green or red fluorescence.

Click-iT™ TUNEL Alexa Fluor™ 594 Imaging Assay, for microscopy & HCS (Invitrogen™)

The Click-iT® TUNEL Alexa Fluor® 594 Imaging Assay utilizes a dUTP modified with an alkyne, a small, bio-orthogonal functional group that enables the nucleotide to be more readily incorporated by TdT than other modified nucleotides, including BrdUTP, biotin-dUTP or fluorescein-dUTP. Detection is based on a click reaction, a copper (I) catalyzed reaction between an azide and alkyne. Click chemistry fills the void when methods such as direct labeling or the use of antibodies are not efficient. The small size of the detection reagent, an Alexa Fluor® azide (MW <~1000) compared to that of an antibody (MW ~150,000) enables facile penetration of complex samples with only mild fixation and permeabilization required.

Compared to other assays using one of the other modified nucleotides, the Click-iT® TUNEL assay is able to detect a higher percentage of apoptotic cells under identical conditions. The assay is also fast and is complete within 2 hours. Furthermore, the Click-iT® TUNEL assay allows multiplexing with surface and intracellular biomarker detection.

More tools for apoptosis detection and measurement >

Click-iT™ Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 488 dye (Invitrogen™)

The Click-iT® Plus TUNEL assay detects apoptotic cells in tissue and cultured cell samples through the use of a small, highly specific labeling moiety and a bright fluorescent dye. After incorporation of the labeling moiety into DNA fragments, detection is achieved through a catalyzed “click” reaction using conditions mild enough to preserve the fluorescent signal from GFP or RFP.

More tools for apoptosis detection and measurement >

• Optimized for the detection of apoptotic cells in either tissue or cell samples
• Multiplex enabled—optimized to work with fluorescent dyes or proteins such as GFP and RFP
• Improved TUNEL assay—better label incorporation due to small reactive moiety
• Bright apoptotic signal—uses Alexa Fluor® dyes, resulting in a stable, non-photobleaching fluorescent signal
• Flexibility—the assay can be configured to test from 1 to 50 samples at a time

Fragmentation of cellular DNA is a hallmark of apoptosis. The TUNEL assay is the most widely used method to detect fragmented DNA in apoptotic cells in tissue samples. The TUNEL assay begins with incorporation of modified dUTP at the 3’-OH end of the fragmented DNA. The dUTP modification is often the addition of a fluorophore. And due to the size of the fluorophore, the modified dUTP can display lower than expected incorporation rates. These lower incorporation rates can affect the sensitivity of the TUNEL assay. Additionally, the fluorophores used in currently available TUNEL assays suffer from photobleaching and fluorescent spectral overlap issues, both of which reduce sensitivity and ability to multiplex the assay.

The Click-iT® Plus TUNEL assay was developed to address these issues. The assay uses dUTP modified with an alkyne group (a small bio-orthogonal functional group) that allows the nucleotide to be more readily incorporated. After incorporation, a highly specific click reaction between the alkyne group and an Alexa Fluor® picolyl azide fluorescent dye, and subsequent detection of that dye, results in a sensitive and specific assay for the detection of apoptotic cells. Because of the gentle reaction conditions, the Click-iT® Plus TUNEL assay enables multiplexing with fluorescent proteins or dyes.

The Click-iT® Plus TUNEL assay has been validated with several different formalin-fixed paraffin-embedded tissue types. In all cases the ability to multiplex with fluorescent proteins and dyes was preserved. Additionally, the ability to stain actin using fluorescent-labeled phalloidin was also preserved.

The Click-iT® Plus TUNEL assay contains all of the reagents needed to detect apoptotic cells from either tissue or cell samples. The reagents supplied in this kit can be used to test 50 samples and can be configured to test from 1 to 50 samples at a time.

Image-iT™ LIVE Green Caspase-3 and -7 Detection Kit, for microscopy (Invitrogen™)

The Image-iT™ LIVE Green Caspase-3 and -7 Detection Kit employs a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA™) methodology, essentially an affinity label. The reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amine acid sequence. For caspase-3 and -7, this recognition sequence is aspartic acid-glutamic acid-valine-aspartic acid (DEVD). A fluorescein group is attached as a reporter. The FLICA reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The FLICA inhibitor is cell permeant and noncytotoxic. Unbound FLICA molecules diffuse out of the cell and are washed away; the remaining green-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added.

Dead Cell Apoptosis Kit with Annexin V FITC and PI, for flow cytometry (Invitrogen™)

This product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to green-fluorescent FITC dye and dead cells using propidium iodide (PI). Propidium iodide stains necrotic cells with red fluorescence. After treatment with both probes, apoptotic cells show green fluorescence, dead cells show red and green fluorescence, and live cells show little or no fluorescence.

View a selection guide for all apoptosis assays for flow cytometry.

CaspGLOW™ Fluorescein Active Caspase-9 Staining Kit (Invitrogen™)

The CaspGLOW™ Fluorescein Active Caspase-9 Staining Kit contains all the reagents necessary to detect active caspase-9 in mammalian cells with high sensitivity. Fluorescein (FITC)-conjugated LEHD-FMK, a specific inhibitor of caspase-9, is utilized in this assay for detection. This reagent is cell permeable, non-toxic and irreversibly binds to the active enzyme. Detection of the labeled cells can be determined by flow cytometry, fluorescent microscopy or a fluorescent plate reader.

The caspases constitute a family of aspartate-specific cysteine proteases that mediate a sequence of cleavage events. Recruitment of the inactive proenzyme to oligomerized receptors leads to caspase activation and autoproteolytic cleavage. These active enzymes can then cleave other caspases, thereby generating a caspase signaling cascade that leads to a form of programmed cell death termed apoptosis.

Caspase-9, which is also known as ICE-LAP6 and Mch6, cleaves its substrates at the C-terminal aspartic acid residue of the motif Asp-X-X-Asp. Activation of this signaling protease occurs upon stimulation of death receptors such as CD95/APO-1/Fas, TRAIL-R1, TRAIL-R2, TNFR1, and TRAMP. Caspase-9 is activated by recruitment and dimerization within the Apaf-1 apoptosome. Once recruited, caspase-9 undergoes proteolytic cleavage at Asp315 to yield 35-kDa and 10-kDa fragments. Unlike other caspases, this cleavage event is not required for the catalytic activity of caspase-9. As an initiator caspase, this protease initiates a caspase signaling cascade that results in apoptosis. Substrates of caspase-9 include caspases-3, -6, and -7. Finally, dimerization of cleaved caspase-9 is inhibited by X-linked inhibitor of apoptosis (XIAP).

Reported Application
Flow Cytometric Analysis, Immunocytochemistry

Vybrant™ FAM Poly Caspases Assay Kit, for flow cytometry (Invitrogen™)

The Vybrant™ FAM Poly Caspases Assay Kit employs a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA™) methodology, essentially an affinity label. The reagent associates a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, with a caspase-specific amine acid sequence. For poly caspases, this recognition sequence is valine-alanine-aspartic acid (VAD). A fluorescein group is attached as a reporter. The FLICA reagent is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently through the FMK moiety. The FLICA inhibitor is cell permeant and noncytotoxic. Unbound FLICA molecules diffuse out of the cell and are washed away; the remaining green-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added. This kit includes the FAM-VAD-FMK FLICA reagent, Hoechst 33342 stain, and propidium iodide stain, which allows the simultaneous evaluation of caspase activation, membrane permeability, and cell cycle by flow cytometry.

View a selection guide for all apoptosis assays for flow cytometry.

Membrane Permeability Dead Cell Apoptosis Kit with PO-PRO™-1 and 7-Aminoactinomycin D, for flow cytometry (Invitrogen™)

This flow cytometry product detects apoptosis on the basis of changes that occur in the permeability of cell membranes using PO-PRO™-1 and 7-aminoactinomycin D (7-AAD) nucleic acid stains. After staining a cell population with PO-PRO™-1 dye and 7-AAD, apoptotic cells show violet fluorescence, dead cells show violet and red fluorescence, and live cells show little or no fluorescence. These populations can easily be distinguished by a flow cytometer that uses both a violet laser and the 488 nm line of an argon-ion laser for excitation. Each kit contains sufficient reagents for approximately 200 flow cytometry tests.

View a selection guide for all apoptosis assays for flow cytometry.

Apoptotic DNA Ladder Kit (Invitrogen™)

The ApoTarget™ Quick Apoptotic DNA Ladder Detection Kit provides a simple and rapid procedure for extraction of chromosomal DNA. The procedure prepares DNA for use in the methods that detect DNA fragmentation in apoptotic cells. Unlike other kits requiring 1 to 2 days to finish, this detection method requires only less than 90 minutes to prepare DNA in a single tube without the need for extraction or column steps. DNA fragmentation can be easily visualized by agarose gel electrophoresis. This procedure increases recovery of small fragmented DNA, thereby improving the sensitivity of the assay.

APO-BrdU™ TUNEL Assay Kit, with Alexa Fluor™ 488 Anti-BrdU (Invitrogen™)

In collaboration with Phoenix Flow Systems, we offer the APO™-BrdU TUNEL Assay Kit which provides all the materials necessary to label and detect the DNA strand breaks of apoptotic cells. When DNA strands are cleaved or nicked by nucleases, a large number of 3´hydroxyl ends are exposed. In the APO-BrdU assay, these ends are labeled with BrdUTP and terminal deoxynucleotidyl transferase (TdT) using the TUNEL technique. Once incorporated ino the DNA, BrdU is detected using a bright and photostable green-fluorescent Alexa Fluor™ 488 dye-labeled anti-BrdU antibody.

View a selection guide for all apoptosis assays for flow cytometry.

Mitochondrial Membrane Potential Apoptosis Kit, with Mitotracker™ Red & Annexin V Alexa Fluor™ 488, for flow cytometry (Invitrogen™)

This flow cytometry product provides a rapid and convenient assay for two hallmarks of apoptosis - phosphatidylserine externalization and changes in mitochondrial membrane potential. After staining a cell population with Alexa Fluor™ 488 annexin V and MitoTracker™ Red CMXRos dye in the provided binding buffer, live cells exhibit very little green fluorescence and bright red fluorescence, whereas apoptotic cells exhibit green fluorescence and decreased red fluorescence. These populations can easily be distinguished using a flow cytometer, and the 488 nm line of an argon-ion laser can be used to excite both dyes.

View a selection guide for all apoptosis assays for flow cytometry.

APO BrdU (TUNEL) Kit (Invitrogen™)

The ApoTarget™ APO-BRDU Kit is a two-color staining method for labeling DNA breaks and total cellular DNA to detect apoptotic cells by flow cytometry.

View a selection guide for all apoptosis assays for flow cytometry.

Single-Channel Dead Cell Apoptosis Kit with Annexin V Alexa Fluor™ 488 and SYTOX™ Green Dyes, for flow cytometry (Invitrogen™)

This product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to green-fluorescent Alexa Fluor™ 488 dye and dead cells using SYTOX™ Green nucleic acid stain. After treatment with both probes, apoptotic cells show green fluorescence, dead cells show brighter green fluorescence, and live cells show little or no fluorescence. These populations can easily be distinguished in the FL1 channel of a flow cytometer, freeing the other channels for detection of other fluorescent colors.

View a selection guide for all apoptosis assays for flow cytometry.

Metabolic Activity Dead Cell Apoptosis Kit with C12 Resazurin, Annexin V APC, and SYTOX™ Green, for flow cytometry (Invitrogen™)

This flow cytometry product provides a three-color fluorescence assay that distinguishes live, apoptotic, and late apoptotic cells from one another. These populations can easily be distinguished using a flow cytometer that has both 488 nm and 633 nm excitation sources (an argon-ion laser and a HeNe laser) and the following reagents Annexin V to detect phosphatidylserine, C12 resazurin for cell metabolism, and SYTOX™ Green nucleic acid stain for compromised membranes.

View a selection guide for all apoptosis assays for flow cytometry.

Dead Cell Apoptosis Kit with Annexin V APC and SYTOX™ Green, for flow cytometry (Invitrogen™)

This flow cytometry product detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to red laser-excited allophycocyanin, and dead cells using SYTOX™ Green nucleic acid stain. Apoptotic cells are detected by annexin V binding to externalized phosphatidylserine, and late apoptotic and necrotic cells have compromised membranes that permit SYTOX™ Green stain access to cellular nucleic acids. After exposing a cell population to APC annexin V and SYTOX™ Green stain, live cells show little or no fluorescence, apoptotic cells show red fluorescence and very little green fluorescence, and late apoptotic cells show a higher level of red and orange fluorescence. These populations can easily be distinguished using a flow cytometer that has both 488 nm and 633 nm excitation sources (an argon-ion laser and a HeNe laser).

View a selection guide for all apoptosis assays for flow cytometry.