Shop All Apoptosis Kits

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.

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.

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-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

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-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

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.

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.

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 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.

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.

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.

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

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.