Shop All Apoptosis Kits

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.

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

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.

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.

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.

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.

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.

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™ 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.

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