Shop All Apoptosis Kits

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.

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.

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.

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.

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

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.

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.

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.

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

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

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.

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.

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.

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.

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

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.

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

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

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 >

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

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.

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.

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.

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

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

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.

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

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.

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.

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• 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.

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