Shop All Apoptosis Kits

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.

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.

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

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.

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.

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

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.

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.

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 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.

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.

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

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.

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.

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.