Assays for Apoptosis - Section 15.5

DNA Stains for Detecting Apoptotic Cells

The characteristic breakdown of the nucleus during apoptosis comprises collapse and fragmentation of the chromatin, degradation of the nuclear envelope and nuclear blebbing, resulting in the formation of micronuclei. Therefore, nucleic acid stains can be useful tools for identifying even low numbers of apoptotic cells in cell populations. Several nucleic acid stains, all of which are listed in Nucleic Acid Stains - Section 8.1, have been used to detect apoptotic cells by fluorescence imaging or flow cytometry.

• Our YO-PRO-1 (Y3603) nucleic acid stain is the basis of an important assay for apoptotic cells that is compatible with both fluorescence microscopy and flow cytometry. Selective uptake of YO-PRO-1 by apoptotic cells of a dexamethasone-treated population of thymocytes, an irradiated peripheral blood mononuclear cell population and a growth factor–depleted tumor B cell line was confirmed by cell sorting. Unlike Hoechst 33342 staining, YO-PRO-1 staining had no effect on the ability of stained T cells to proliferate. Moreover, the visible-light absorption of the YO-PRO-1 stain () eliminates the need for UV excitation capabilities in flow cytometry. YO-PRO-1 is the key reagent in our Vybrant Apoptosis Assay Kits #4 and #7 (V13243, V23201, see below), which provide the reagents and tested protocols for combination flow cytometric apoptosis and necrosis assays.
• Some of our cell-permeant, green-fluorescent SYTO dyes, including the SYTO 13 and SYTO 16 nucleic acid stains (S7575, S7578), are proving useful for distinguishing apoptotic neuronal cells and apoptotic thymocytes. Our SYTO Fluorescent Nucleic Acid Stain Sampler Kits (S7554, S7572, S11340, S11350, S11360; Nucleic Acid Stains - Section 8.1) provide fluorescent SYTO dyes covering the entire visible spectrum (Cell-permeant cyanine nucleic acid stains - Table 8.3) that may be screened for their utility in monitoring apoptosis. In addition, apoptotic cells in a follicular lymphoma cell line could be discriminated earlier with our SYTO 17 red-fluorescent nucleic acid stain (S7579) than with either fluorescein-labeled annexin V or propidium iodide.
• Hoechst 33342 (H1399, H3570; FluoroPure Grade - Note 19.2, H21492) is readily taken up by cells during the initial stages of apoptosis, whereas cell-impermeant dyes such as propidium iodide (P1304MP, P3566, P21493; Nucleic Acid Stains - Section 8.1) and ethidium bromide (E1305, E3565; Nucleic Acid Stains - Section 8.1) are excluded. Later stages of apoptosis are accompanied by an increase in membrane permeability, which allows propidium iodide to enter cells. Thus, a combination of Hoechst 33342 and propidium iodide has been extensively used for simultaneous flow cytometric and fluorescence imaging analysis of the stages of apoptosis and cell-cycle distribution. Our Vybrant Apoptosis Assay Kit #5 (V13244, see below) is based on these reagents, and our Vybrant Apoptosis Assay Kit #7 (V23201, see below) adds the YO-PRO-1 nucleic acid to selectively determine the apoptotic cell population in a three-color experiment.
• The rate of Hoechst 33342 uptake in partially apoptotic cell populations is correlated with low intracellular pH, as measured with our carboxy SNARF-1 pH indicator (C1271, C1272; Probes Useful at Near-Neutral pH - Section 20.2).
• Hoechst 33342, which selectively stains nuclei of apoptotic cells blue fluorescent, has also been used in combination with calcein AM (C1430, C3099, C3100MP; Viability and Cytotoxicity Assay Reagents - Section 15.2), which stains all cells that have intact membranes — even apoptotic cells — green fluorescent. Presumably the dead-cell population could be selectively detected using propidium iodide to make this a three-color assay.
• 7-Aminoactinomycin D (7-AAD, A1310) has been used alone or in combination with Hoechst 33342 to separate populations of live cells, early apoptotic cells and late apoptotic cells by flow cytometry. The staining pattern of 7-AAD is retained following cell fixation, and its unusually large Stokes shift is advantageous when simultaneously staining with cell-surface labels. 7-AAD staining has also been used to detect apoptotic cells by their characteristic morphology using fluorescence microscopy. 7-AAD has also been used in combination with the green-fluorescent SYTO 16 nucleic acid stain (S7578) to detect early stages of apoptosis that could not be detected by 7-AAD alone.
• The cell-permeant nucleic acid stain LDS 751 (L7595) has been used to discriminate intact nucleated cells from nonnucleated cells and cells with damaged nuclei, as well as to differentiate apoptotic cells from nonapoptotic cells.
• Acridine orange (A1301, A3568) exhibits metachromatic fluorescence that is sensitive to DNA conformation, making it a useful probe for detecting apoptotic cells. When analyzed by flow cytometry, apoptotic cells stained by acridine orange show reduced green fluorescence and enhanced red fluorescence in comparison to normal cells.
• DAPI (D1306, D21490; Nucleic Acid Stains - Section 8.1) and sulforhodamine 101 (S359, Polar Tracers - Section 14.3) can be used together in fixed apoptotic cells to reveal concomitant breakdown of proteins and DNA.
• The excited-state lifetime of ethidium homodimer-2 (E3599, Nucleic Acid Stains - Section 8.1) has been shown to be different in populations of aldehyde-fixed apoptotic and nonapoptotic cells.
• Ethidium monoazide (E1374, Viability and Cytotoxicity Assay Reagents - Section 15.2) passes through the partially compromised membrane of apoptotic cells; photolysis results in covalent labeling of intracellular nucleic acids that persists through fixation and permeabilization.

DNA fragmentation can also be detected in vitro using electrophoresis. DNA extracted from apoptotic cells, separated by gel electrophoresis and stained with ethidium bromide reveals a characteristic ladder pattern of low molecular weight DNA fragments. Ethidium bromide has been used for a dot-blot assay to detect apoptotic DNA fragments. Our ultrasensitive SYBR Green I nucleic acid stain (S7567, Nucleic Acid Detection and Quantitation in Electrophoretic Gels and Capillaries - Section 8.4) and SYBR DX DNA blot stain (S7550, Detecting Nucleic Acid Hybridization - Section 8.5) allow the detection of even fewer apoptotic cells in these applications (). Electrophoresis of apoptotic cells in an agarose gel matrix results in the formation of distinctive "comets" of DNA leaking from apoptotic cells (but not normal cells; see the paragraph, Comet (Single-Cell Gel Electrophoresis) Assay to Detect Damaged DNA, below) ().

Vybrant Apoptosis Assay Kit #4

Our Vybrant Apoptosis Assay Kit #4 (V13243) detects apoptosis based on changes that occur in the permeability of cell membranes. This kit contains ready-to-use solutions of both the YO-PRO-1 and propidium iodide nucleic acid stains. Our Patented YO-PRO-1 nucleic acid stain selectively passes through the plasma membranes of apoptotic cells and labels them with moderate green fluorescence. Necrotic cells are stained with the red-fluorescent propidium iodide, a DNA-selective dye that is membrane impermeant but that easily passes through the compromised plasma membranes of necrotic cells. Live cells are not appreciably stained by either YO-PRO-1 or propidium iodide. The dyes included in the Vybrant Apoptosis Assay Kit #4 are effectively excited by the 488 nm spectral line of the argon-ion laser and are useful for both flow cytometry (Figure 15.85) and fluorescence microscopy (). We have optimized our Vybrant Apoptosis Assay Kits using Jurkat cells, a human T-cell leukemia clone, treated with camptothecin to induce apoptosis. Some modifications may be required for use with other cell types. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Figure 15.85 Flow cytometric analysis of Jurkat cells using the Vybrant Apoptosis Assay Kit #4 (V13243). Jurkat human T-cell leukemia cells were first exposed to10 µM camptothecin for four hours (top panel) or left untreated (as control, bottom panel). Cells were then treated with the reagents in the Vybrant Apoptosis Assay Kit #4 and analyzed by flow cytometry. Note that the camptothecin-treated cells (top panel) have a significantly higher percentage of apoptotic cells (indicated by an "A") than the basal level of apoptosis seen in the control cells (bottom panel). V = viable cells, D = dead cells.

Vybrant Apoptosis Assay Kit #5

The Vybrant Apoptosis Assay Kit #5 (V13244) provides a rapid and convenient assay for apoptosis based upon fluorescence detection of the compacted state of the chromatin in apoptotic cells. This kit contains ready-to-use solutions of the blue-fluorescent Hoechst 33342 dye (excitation/emission maxima ~350/461 nm when bound to DNA), which stains the condensed chromatin of apoptotic cells more brightly than the chromatin of nonapoptotic cells, and the red-fluorescent propidium iodide (excitation/emission maxima ~535/617 nm when bound to DNA), which is permeant only to dead cells with compromised membranes. The staining pattern resulting from the simultaneous use of these dyes makes it possible to distinguish normal, apoptotic and dead cell populations by flow cytometry or fluorescence microscopy. The 351 nm spectral line of an argon-ion laser or other suitable UV source is required for excitation of the Hoechst 33342 dye, whereas propidium iodide can be excited with the 488 nm spectral line of an argon-ion laser. We have optimized this assay using Jurkat cells, a human T-cell leukemia clone, treated with camptothecin to induce apoptosis. Some modifications may be required for use with other cell types. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Vybrant Apoptosis Assay Kit #7

The Vybrant Apoptosis Assay Kit #7 (V23201) combines the detection principles used in our Vybrant Apoptosis Assay Kits #4 and #5 (see above). Three nucleic acid stains — Hoechst 33342, YO-PRO-1 and propidium iodide — are utilized to identify by flow cytometry the blue-fluorescent live-cell population, the green-fluorescent apoptotic population and the red-fluorescent dead-cell population. The stains are provided as separate solutions to facilitate optimization of the assay for the cell line under study and the equipment available. However, once optimized, the assay can be completed using simultaneous staining with a mixture of the three nucleic acid stains and either UV excitation of all three dyes or with a combination of UV excitation for the Hoechst 33342 dye and excitation by the 488 nm spectral line of the argon-ion laser. Differences in the intensity of the dye staining may make it difficult to simultaneously photograph the live, apoptotic and dead cells by microscopy. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Vybrant Apoptosis Assay Kit #12

The Vybrant Apoptosis Assay Kit #12 (V35121) provides a rapid and convenient assay for apoptosis based upon fluorescence detection of the compacted state of the chromatin in apoptotic cells. This kit contains ready-to-use solutions of the violet-fluorescent Vybrant DyeCycle Violet stain (excitation/emission maxima ~370/440 nm when bound to DNA), which stains the condensed chromatin of apoptotic cells more brightly than the chromatin of nonapoptotic cells, and the red-fluorescent 7-aminoactinomycin (7-AAD) (excitation/emission maxima ~546/650 nm when bound to DNA), which is permeant only to dead cells with compromised membranes. The staining pattern resulting from the simultaneous use of these dyes makes it possible to distinguish normal, apoptotic and dead cell populations by flow cytometry. The 405 nm spectral line of the violet laser is used for excitation of the Vybrant DyeCycle Violet stain, whereas 7-AAD may be excited by the 488 nm spectral line of an argon-ion laser. We have optimized this assay using Jurkat cells, a human T-cell leukemia clone, treated with camptothecin to induce apoptosis. Some modifications may be required for use with other cell types. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Vybrant Apoptosis Assay Kit #13

Like the Vybrant Apoptosis Assay Kit #4, the Vybrant Apoptosis Assay Kit #13 (V35123) detects apoptosis based on changes that occur in the permeability of cell membranes (Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4). This kit contains ready-to-use solutions of both PO-PRO-1 and 7-aminoactinomycin (7-AAD) nucleic acid stains. Our Patented PO-PRO-1 nucleic acid stain selectively passes through the plasma membranes of apoptotic cells and labels them with violet fluorescence. Furthermore, annexin V labeling of apoptosis yields poor results with trypsinized cells, whereas PO-PRO-1 dye provides te same efficiency for detecting apoptosis with trypsinized cells as it does with suspension cells. Necrotic cells are stained with the red-fluorescent 7-AAD, a DNA-selective dye that is membrane impermeant but that easily passes through the compromised plasma membranes of necrotic cells. Live cells are not appreciably stained by either PO-PRO-1 or 7-AAD. The dyes included in the Vybrant Apoptosis Assay Kit #13 are effectively excited by a flow cytometer that uses both the 405 nm spectral line of the violet laser and the 488 nm spectral line of the argon-ion laser for excitation. We have optimized our Vybrant Apoptosis Assay Kits using Jurkat cells, a human T-cell leukemia clone, treated with camptothecin to induce apoptosis. Some modifications may be required for use with other cell types. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Comet (Single-Cell Gel Electrophoresis) Assay to Detect Damaged DNA

The Comet assay, or single-cell gel electrophoresis assay, is used for rapid detection and quantitation of DNA damage from single cells. The Comet assay is based on the alkaline lysis of labile DNA at sites of damage. Cells are immobilized in a thin agarose matrix on slides and gently lysed. When subjected to electrophoresis, the unwound, relaxed DNA migrates out of the cells. After staining with a nucleic acid stain, cells that have accumulated DNA damage appear as fluorescent comets, with tails of DNA fragmentation or unwinding (). In contrast, cells with normal, undamaged DNA appear as round dots, because their intact DNA does not migrate out of the cell. The ease and sensitivity of the Comet assay has provided a fast and convenient way to measure damage to human sperm DNA, evaluate DNA replicative integrity, monitor the sensitivity of tumor cells to radiation damage and assess the sensitivity of molluscan cells to toxins in the environment. The Comet assay can also be used in combination with FISH (Detecting Nucleic Acid Hybridization - Section 8.5) to identify specific sequences with damaged DNA.

Comet assays have traditionally been performed using ethidium bromide to stain the DNA. However, our YOYO-1 dye was found to increase the sensitivity of the assay eightfold, as compared with ethidium bromide. Use of the SYBR Gold and SYBR Green I stains improves the sensitivity of this assay ().

DNA fragmentation that occurs during apoptosis produces DNA strand breaks. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assays are widely used for detecting DNA nicks in apoptotic cells. Once the cells are fixed, DNA strand breaks can be detected in situ using mammalian terminal deoxynucleotidyl transferase (TdT), which covalently adds labeled nucleotides to the 3'-hydroxyl ends of these DNA fragments in a template-independent fashion. Break sites have traditionally been labeled with biotinylated dUTP, followed by subsequent detection with an avidin or streptavidin conjugate (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23). However, a more direct approach for detecting DNA strand breaks in apoptotic cells is possible via the use of our ChromaTide BODIPY FL-14-dUTP (C7614) as a TdT substrate ().

The single-step BODIPY FL dye–based assay has several advantages over indirect detection of biotinylated or haptenylated nucleotides, including fewer protocol steps and increased cell yields. BODIPY FL dye–labeled nucleotides have also proven superior to fluorescein-labeled nucleotides for detection of DNA strand breaks in apoptotic cells because they provide stronger signals, a narrower emission spectrum and less photobleaching (). Moreover, it has been reported that ChromaTide BODIPY FL-14-dUTP incorporated into the granules of the condensed chromatin structure of late-apoptotic cells — cells characterized by extensive nuclear fragmentation — exhibits yellow fluorescence, whereas uncondensed areas of the nuclei or early-apoptotic cells exhibit green fluorescence. This spectral shift, which is characteristic of the BODIPY fluorophores, is most likely a consequence of stacking of the BODIPY FL fluorophores (Figure 13.6) and could be very useful for identifying the stages of apoptosis on a single-cell basis. Our ChromaTide Texas Red-12-dUTP (C7631) has been used similarly for a TdT-mediated apoptosis assay. Presumably a number of the ChromaTide dUTP nucleotides listed in Characteristics of ChromaTide dUTP, ChromaTide OBEA-dCTP, aha-dUTP and aha-dCTP labeled nucleotides - Table 8.7 could be used for the direct or indirect TUNEL assay; we have not yet tried the ChromaTide dCTP nucleotides in this assay. Furthermore, our anti-dye antibodies (Anti-Dye and Anti-Hapten Antibodies - Section 7.4) can amplify the signal of many of the dyes used to prepare the ChromaTide nucleotides.

Figure 13.6 A) Fluorescence spectrum of β-C₈-BODIPY 500/510 C₅-HPC (D3795) incorporated in DOPC (dioctadecenoylphosphocholine) liposomes at 1:100 mole:mole (labeled:unlabeled PC). B) Fluorescence spectra at high molar incorporation levels: 1:10 mole:mole and 1:5 mole:mole.

In situ DNA modifications by labeled nucleotides have been used to detect DNA fragmentation in what may be apoptotic cells in autopsy brains of Huntington's and Alzheimer's disease patients. DNA fragmentation is also associated with amyotrophic lateral sclerosis. Analogous to TdT's ability to label double-strand breaks, the E. coli repair enzyme DNA polymerase I can be used to detect single-strand nicks, which appear as a relatively early step in some apoptotic processes. Because our ChromaTide BODIPY FL-14-dUTP (C7614) and ChromaTide fluorescein-12-dUTP (C7604) are incorporated into DNA by E. coli DNA polymerase I, it is likely that they may also be effective for in situ labeling with the nick translation method.

Because DNA fragmentation is one of the most reliable methods for detecting apoptosis, we have collaborated with Phoenix Flow Systems to offer the APO-BrdU TUNEL Assay Kit (A23210), which provides all the materials necessary to label and detect the DNA strand breaks of apoptotic cells. When DNA strands are cleaved or nicked by nucleases, a large number of 3'-hydroxyl ends are exposed. In the APO-BrdU assay, these ends are labeled with BrdUTP and terminal deoxynucleotidyl transferase (TdT) using the TUNEL technique described above. Once incorporated into the DNA, BrdU is detected using an Alexa Fluor 488 dye–labeled anti-BrdU monoclonal antibody (). This kit also provides propidium iodide for determining total cellular DNA content, as well as fixed control cells for assessing assay performance.

The APO-BrdU TUNEL Assay Kit includes complete protocols for use in flow cytometry applications, though it may also be adapted for use with fluorescence microscopy. Each kit contains:

• Terminal deoxynucleotidyl transferase (TdT), for catalyzing the addition of BrdUTP at the break sites
• 5-Bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP)
• Alexa Fluor 488 dye–labeled anti-BrdU mouse monoclonal antibody PRB-1, for detecting BrdU labels
• Propidium iodide/RNase staining buffer, for quantitating total cellular DNA
• Reaction, wash and rinse buffers
• Positive control cells (a fixed human lymphoma cell line)
• Negative control cells (a fixed human lymphoma cell line)
• Detailed protocols (APO-BrdU TUNEL Assay Kit)

Sufficient reagents are provided for approximately 60 assays of 1 mL samples, each containing 1–2 × 10⁶ cells/mL.

Annexin V Conjugates

Molecular Probes is collaborating with Nexins Research BV — the original developer of fluorescent phosphatidylserine-binding proteins — to provide what we feel are the best and brightest annexin V conjugates available. The human vascular anticoagulant annexin V is a 35–36 kilodalton, Ca²⁺-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine (PS). In normal viable cells, PS is located on the cytoplasmic surface of the cell membrane. However, in apoptotic cells, PS is translocated from the inner to the outer leaflet of the plasma membrane, exposing PS to the external cellular environment where it can be detected by annexin V conjugates. In leukocyte apoptosis, PS on the outer surface of the cell marks the cell for recognition and phagocytosis by macrophages.

Highly fluorescent annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine, an indicator of intermediate stages of apoptosis. Nuclear fragmentation, mitochondrial membrane potential flux and caspase-3 activation apparently precede phosphatidylserine "flipping" during apoptosis, whereas permeability to propidium iodide and cytoskeletal collapse occur later. The difference in fluorescence intensity between apoptotic and nonapoptotic cells stained by our fluorescent annexin V conjugates, as measured by flow cytometry, is typically about 100-fold (Figure 15.90). Annexin V conjugates (Annexin V Conjugates for Apoptosis Detection) are very useful for flow cytometry, confocal or epifluorescence microscopy and, like antibody staining, can be used in combination with other dyes, including nucleic acid stains, to accurately assess mixed populations of apoptotic and nonapoptotic cells. Our annexin V conjugates are available as stand-alone reagents, each suitable for 50–100 flow cytometry assays or many more imaging assays, or in several variations of our Vybrant Apoptosis Assay Kits (Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4). Our annexin V conjugates include:

• Alexa Fluor 488 annexin V (A13201, ), a green-fluorescent conjugate (excitation/emission maxima ~495/519 nm) that has spectral characteristics similar to fluorescein conjugates, but exhibits fluorescence that is brighter, much more photostable and less pH dependent (Figure 1.9, , , Figure 1.53). Alexa Fluor 488 annexin V is used in both our Vybrant Apoptosis Assays Kits #1 and #2 (V13240, V13241; see below), which contain all of the reagents and an easy-to-follow protocol for flow cytometric detection and quantitation of apoptotic cells.
• Fluorescein (FITC) annexin V (A13199), a green-fluorescent conjugate that has been extensively used by a number of laboratories to detect apoptotic cells populations. Fluorescein annexin V is frequently used in combination with propidium iodide to detect necrotic cells, as in our Vybrant Apoptosis Assay Kit #3 (V13242, see below).
• Oregon Green 488 annexin V (A13200), a green-fluorescent conjugate that is spectrally similar to the fluorescein annexin V conjugate but is brighter and more photostable (Figure 1.46).
• R-phycoerythrin annexin V (A35111), a highly fluorescent phycobiliprotein conjugate with absorption maxima at 496 nm, 546 nm and 565 nm and an emission maximum at 578 nm.
• Alexa Fluor 568 annexin V (A13202), a red-orange–fluorescent annexin V conjugate (excitation/emission maxima ~578/603 nm) with exceptionally bright and photostable fluorescence. We have determined that this conjugate can be used for simultaneous staining with green-fluorescent probes, such as our green-fluorescent Alexa Fluor 488 anti–CD 4 conjugate (A21335, Primary Antibodies for Diverse Applications - Section 7.5), for multiparameter experiments.
• Alexa Fluor 594 annexin V (A13203), a red-fluorescent annexin V conjugate with spectra similar to those of Texas Red conjugates (excitation/emission maxima ~590/617 nm) that can be used with green-fluorescent probes for multiparameter experiments. The Alexa Fluor 594 conjugate is readily excited by the 568 nm spectral line used in many confocal laser-scanning microscopes and has fluorescence that is well separated from the emission of green-fluorescent probes.
• Alexa Fluor 647 annexin V (A23204), which permits use of long-wavelength excitation sources for detection of apoptotic cells by either flow cytometry or microscopy.
• Allophycocyanin annexin V (A35110), a highly fluorescent phycobiliprotein conjugate with absorption/emission maxima of 650/660 nm.
• Alexa Fluor 350 annexin V (A23202) can be excited in the ultraviolet and has bright blue fluorescence. Alternatively, the reagents in our Vybrant Apoptosis Assay Kit #6 (V23200) can be used in this spectral region.
• Pacific Blue annexin V (A35122) can be excited with the 405 nm spectral line of the violet laser, making it ideal for instruments equipped with a violet laser and for multicolor experiments that include green- or red-fluorescent dyes.
  
• Biotin-X annexin V (A13204), which can be detected by any of our fluorescent avidin or streptavidin conjugates (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6, Molecular Probes' selection of avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates - Table 7.23), gives the researcher the ultimate in color selection for multiparameter experiments. Biotin-X annexin V also permits detection of apoptotic cells by electron microscopy and should permit separation of apoptotic cells with our Captivate ferrofluid streptavidin conjugate (C21476, Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices - Section 7.6), optionally in combination with the Captivate microscope-mounted magnetic yoke assembly (C24700, Accessories for Fluorescence Microscopy and Magnetic Separation - Section 23.3, Figure).

Vybrant Apoptosis Assay Kit #1

With the Vybrant Apoptosis Assay Kit #1 (V13240), apoptotic cells are detected based on the externalization of phosphatidylserine. This kit contains recombinant annexin V conjugated to the Alexa Fluor 488 dye, our brightest and most photostable green fluorophore, to provide maximum sensitivity. In addition, the kit includes a ready-to-use solution of the SYTOX Green nucleic acid stain. The SYTOX Green dye is impermeant to live cells and apoptotic cells but stains necrotic cells with intense green fluorescence by binding to cellular nucleic acids. After staining a cell population with Alexa Fluor 488 annexin V and SYTOX Green dye in the provided binding buffer, apoptotic cells show green fluorescence, dead cells show a higher level of green fluorescence and live cells show little or no fluorescence (Figure 15.92). These populations can easily be distinguished using a flow cytometer with the 488 nm spectral line of an argon-ion laser for excitation. Both Alexa Fluor 488 annexin and the SYTOX Green dye emit a green fluorescence that can be detected in the green channel, freeing the other channels for the detection of additional probes in multicolor labeling experiments. We have optimized the Vybrant Apoptosis Assay Kits using Jurkat cells, a human T-cell leukemia clone, treated with camptothecin to induce apoptosis. Some modifications may be required for use with other cell types. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Figure 15.92 Flow cytometric analysis of Jurkat cells using the Vybrant Apoptosis Assay Kit #1 (V13240). Jurkat human T-cell leukemia cells were first exposed to 10 µM camptothecin for four hours green line) or left untreated (as control, blue line). Cells were then treated with the reagents in the Vybrant Apoptosis Assay Kit #1 and analyzed by flow cytometry. Note that the camptothecin-treated cells (green line) have a significantly higher percentage of apoptotic cells (intermediate green fluorescence) than the basal level of apoptosis seen in the control cells (blue line).

Vybrant Apoptosis Assay Kit #2

Like the Vybrant Apoptosis Kit #1, our Vybrant Apoptosis Assay Kit #2 (V13241) detects the externalization of phosphatidylserine in apoptotic cells. The Vybrant Apoptosis Assay Kit #2 provides a sensitive two-color assay that employs our green-fluorescent Alexa Fluor 488 annexin and a ready-to-use solution of the red-fluorescent propidium iodide nucleic acid stain. Propidium iodide is impermeant to live cells and apoptotic cells but stains necrotic cells with red fluorescence, binding tightly to the nucleic acids in the cell. After staining a cell population with Alexa Fluor 488 annexin V and propidium iodide in the provided binding buffer, apoptotic cells show green fluorescence, dead cells show red and green fluorescence, and live cells show little or no fluorescence (Figure 15.90). These populations can easily be distinguished using a flow cytometer with the 488 nm spectral line of an argon-ion laser for excitation. We have optimized this assay using Jurkat cells, a human T-cell leukemia clone, treated with camptothecin to induce apoptosis. Some modifications may be required for use with other cell types. The Vybrant Apoptosis Assay Kit #2 is designed for use with either flow cytometers or fluorescence microscopes. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Figure 15.90 Flow cytometric analysis of Jurkat cells using the Vybrant Apoptosis Assay Kit #2 (V13241). Jurkat human T-cell leukemia cells were first exposed to 10 µM camptothecin for four hours (bottom panel) or left untreated (as control, top panel). Cells were then treated with the reagents in the Vybrant Apoptosis Assay Kit #2 and analyzed by flow cytometry. Note that the camptothecin-treated cells (bottom panel) have a significantly higher percentage of apoptotic cells (indicated by an "A") than the basal level of apoptosis seen in the control cells (top panel). V = viable cells, D = dead cells.

Vybrant Apoptosis Assay Kit #3

The Vybrant Apoptosis Assay Kit #3 (V13242) is very similar to the Vybrant Apoptosis Assay Kit #2, except that it contains fluorescein (FITC) annexin V in place of the Alexa Fluor 488 conjugate found in Kit #2. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Vybrant Apoptosis Assay Kit #6

The Vybrant Apoptosis Assay Kit #6 (V23200) is very similar to the Vybrant Apoptosis Assay Kit #2, except that it contains biotin-X annexin V and Alexa Fluor 350 streptavidin in place of the Alexa Fluor 488 conjugate found in Kit #2. After staining a cell population with biotin-X annexin V in the provided binding buffer, Alexa Fluor 350 streptavidin is added to fluorescently label the bound annexin V. Finally, propidium iodide is added to detect necrotic cells. Apoptotic cells show blue fluorescence, dead cells show red and blue fluorescence and live cells show little or no fluorescence. These populations can easily be distinguished using a flow cytometer with UV excitation for the Alexa Fluor 350 fluorophore and 488 nm excitation for the propidium iodide. With the Vybrant Apoptosis Assay Kit #6, fluorescence in the green channel is minimal. In the same experiment for apoptosis detection, the researcher can apply a green-fluorescent probe, for example an antibody labeled with the Alexa Fluor 488 dye or with fluorescein. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Vybrant Apoptosis Assay Kits #8 and #9

The Vybrant Apoptosis Assay Kits #8 and #9 (V35112, V35113) are very similar to the Vybrant Apoptosis Assay Kit #1, except that they contain either R-phycoerythrin (R-PE) annexin V (Vybrant Kit #8) or allophycocyanin annexin V (Vybrant Kit #9) instead of Alexa Fluor 488 annexin V. R-PE is an extremely fluorescent phycobiliprotein that can easily be excited with the 488 nm spectral line of the argon-ion laser on a standard flow cytometer and exhibits an emission maximum at 578 nm. Also intensely fluorescent, allophycocyanin can be efficiently excited with the 633 nm spectral line of the He–Ne laser on a standard flow cytometer and exhibits an emission maximum at 660 nm. In addition to the phycobiliprotein-conjugated annexin V, these kits include the SYTOX Green nucleic acid stain, which is impermeant to live cells and apoptotic cells but stains necrotic cells with intense green fluorescence. After staining a cell population with R-PE annexin V and SYTOX Green stain, apoptotic cells show orange fluorescence with very little green fluorescence, late apoptotic cells show a higher level of green and orange fluorescence and live cells show little or no fluorescence (Figure 15.93); these populations can easily be distinguished using a flow cytometer with the 488 nm spectral line of an argon-ion laser for excitation. After staining a cell population with allophycocyanin annexin V and the SYTOX Green stain, apoptotic cells show far-red fluorescence with very little green fluorescence, late apoptotic cells show a higher level of green and far-red fluorescence and live cells show little or no fluorescence (Figure 15.94); these populations can easily be distinguished using a flow cytometer with both the 488 nm spectral line of an argon-ion laser and the 633 nm spectral line of a He–Ne laser for excitation. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Figure 15.94 Flow cytometric analysis of Jurkat cells using the Vybrant Apoptosis Assay Kit #9 (V35113). Jurkat human T-cell leukemia cells were first exposed to 10 µM camptothecin at 37°C, 5% CO₂. The cells were then treated with the reagents in the Vybrant Apoptosis Assay Kit #9 and analyzed by flow cytometry. The SYTOX Green fluorescence versus allophycocyanin (APC) annexin fluorescence dot plot shows resolution of live, apoptotic and dead cell populations.

Vybrant Apoptosis Assay Kit #10

The Vybrant Apoptosis Assay Kit #10 (V35114 is an enhanced version of the Vybrant Apoptosis Assay Kit #9 because it also contains C₁₂-resazurin. Nonfluorescent C₁₂-resazurin is reduced by viable cells to orange-fluorescent C₁₂-resorufin. Resazurin (also called alamarBlue) has been used extensively to detect the metabolic activity of many different cell types, from bacteria to higher eukaryotes. After staining a cell population with allophycocyanin annexin V, C₁₂-resazurin and the SYTOX Green stain, apoptotic cells show far-red fluorescence, intermediate orange fluorescence and no green fluorescence; late apoptotic cells show intense far-red and green fluorescence and little orange fluorescence; live cells show little or no green or far-red fluorescence but significant orange fluorescence (Figure 15.95). These populations can easily be distinguished using a flow cytometer with both the 488 nm spectral line of an argon-ion laser and the 633 nm spectral line of a He–Ne laser for excitation. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Figure 15.95 Flow cytometric analysis of Jurkat cells using the Vybrant Apoptosis Assay Kit #10 (V35114). Jurkat human T-cell leukemia cells were first exposed to either 10 µM camptothecin or 2 mM hydrogen peroxide for 4 hours at 37°C, 5% CO₂. The cells were then combined, treated with the reagents in the Vybrant Apoptosis Assay Kit #10 and analyzed by flow cytometry. A) The SYTOX Green fluorescence versus allophycocyanin (APC) annexin fluorescence dot plot shows resolution of live, apoptotic and dead cell populations. The cell populations can be evaluated for metabolic activity using B) the dodecylresorufin fluorescence versus SYTOX Green fluorescence dot plot and C) the dodecylresorufin fluorescence versus allophycocyanin fluorescence dot plot.

Vybrant Apoptosis Assay Kit #11

The Vybrant Apoptosis Assay Kit #11 (V35116) provides a rapid and convenient assay for two hallmarks of apoptosis — phosphatidylserine externalization and changes in mitochondrial membrane potential. Recombinant annexin V conjugated to the Alexa Fluor 488 dye, our brightest and most photostable green fluorophore, provides maximum sensitivity for detecting phosphatidylserine externalization in apoptotic cells. Live cells are labeled with MitoTracker Red CMXRos, which exhibits bright red fluorescence in the presence of a mitochondrial transmembrane potential. After staining a cell population with Alexa Fluor 488 annexin V and MitoTracker Red CMXRos dye in the provided binding buffer, live cells exhibit very little green fluorescence and bright red fluorescence, whereas apoptotic cells exhibit bright green fluorescence and decreased red fluorescence (Figure 15.96). These populations can easily be distinguished using a flow cytometer, and the 488 nm line of an argon-ion laser can be used to excite both dyes. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Figure 15.96 Flow cytometric analysis of Jurkat cells using the Vybrant Apoptosis Assay Kit #11 (V35116). Jurkat human T-cell leukemia cells in complete medium were A) first exposed to 10 µM camptothecin for 4 hours or B) left untreated. Both cell populations were then treated with the reagents in the Vybrant Apoptosis Assay Kit #11 and analyzed by flow cytometry. Note that the apoptotic cells show higher reactivity for annexin V and lower MitoTracker Red dye fluorescence than do live cells.

Vybrant Apoptosis Assay Kit #14

Like the Vybrant Apoptosis Kit #1, the Vybrant Apoptosis Assay Kit #14 (V35124) detects the externalization of phosphatidylserine in apoptotic cells. The Vybrant Apoptosis Assay Kit #14 provides a sensitive two-color assay that employs our violet laser–excitable Pacific Blue annexin (excitation/emission maxima ~415/460 nm) and a ready-to-use solution of the red-fluorescent 7-aminoactinomycin (7-AAD) nucleic acid stain (excitation/emission ~546/650 nm). 7-AAD is impermeant to live cells and apoptotic cells but stains necrotic cells with red fluorescence, binding tightly to the nucleic acids in the cell. After staining a cell population with Pacific Blue annexin V and 7-AAD in the provided binding buffer, apoptotic cells show blue fluorescence, dead cells show red fluorescence, and live cells show little or no fluorescence. These populations can easily be distinguished with a flow cytometer that uses both the 405 nm spectral line of the violet laser and the 488 nm spectral line of the argon-ion laser for excitation. We have optimized this assay using Jurkat cells, a human T-cell leukemia clone, treated with camptothecin to induce apoptosis. Some modifications may be required for use with other cell types. The kit components, number of assays and assay principles are summarized in Summary of Molecular Probes' Vybrant Apoptosis Assay Kits - Table 15.4.

Caspase-3 Substrates

A distinctive feature of the early stages of apoptosis is the activation of caspase enzymes. Members of the caspase (CED-3/ICE) family of cysteine–aspartic acid specific proteases have been identified as crucial mediators of the complex biochemical events associated with apoptosis, The recognition site for caspases is marked by three to four amino acids followed by an aspartic acid residue, with the cleavage occurring after the aspartate. The caspase proteases are typically synthesized as inactive precursors. Inhibitor release or cofactor binding activates the caspase through cleavage at internal aspartates, either by autocatalysis or by the action of another protease.

Caspase-3 (CPP32/apopain) is a key effector in the apoptosis pathway, amplifying the signal from initiator caspases (such as caspase-8) and signifying full commitment to cellular disassembly. In addition to cleaving other caspases in the enzyme cascade, caspase-3 has been shown to cleave poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase, protein kinase Cδ and actin. Molecular Probes offers a selection of fluorogenic caspase substrates (Fluorogenic substrates for caspase activity - Table 15.5). The Z-DEVD-AFC substrate, (A22121), which contains containing the caspase-3 recognition site Asp-Glu-Val-Asp (DEVD), undergoes an ~65 nm red-shift to exhibit a peak emission of ~500 nm upon cleavage. The Z-DEVD-R110 substrate, a component of our EnzChek Caspase-3 Assay Kit #2 (E13184) and RediPlate 96 EnzChek Caspase-3 Assay Kit (R35100) described below, is available separately in a 20 mg unit size for high-throughput screening applications (R22120, Rhodamine 110-based bis-peptide substrates - Table 10.2). This nonfluorescent bisamide is first converted by caspase-3 (or a closely related protease) to the fluorescent monoamide and then to the even more fluorescent rhodamine 110 (excitation/emission maxima ~496/520 nm). In addition, the bis-L-aspartic acid amide of R110 (D₂-R110, R22122), which contains rhodamine 110 (R110) flanked by aspartic acid residues, may serve as a substrate for a variety of apoptosis-related proteases, including caspase-3 and caspase-7, and does not appear to require any invasive techniques such as osmotic shock to gain entrance into the cytoplasm ().

Caspase-8 Substrates

Caspase-8 plays a critical role in the early cascade of apoptosis, acting as an initiator of the caspase activation cascade. Activation of the enzyme itself is accomplished through direct interaction with the death domains of cell-surface receptors for apoptosis-inducing ligands. The activated protease has been shown to be involved in a pathway that mediates the release of cytochrome c from the mitochondria and is also known to activate downstream caspases, such as caspase-3. Three fluorogenic substrates containing the caspase-8 recognition sequence Ile-Glu-Thr-Asp (IETD) are available (Fluorogenic substrates for caspase activity - Table 15.5); Z-IETD-AMC and Z-IETD-AFC (A22127, A22128; blue fluorescent after cleavage) and Z-IETD-R110 (R22125, R22126; green fluorescent after cleavage).

Other Caspase and Granzyme B Substrates

In addition to our R110-derived caspase-3 and -8 substrates, we offer R110-based substrates for caspase-1, -2, -6, -9 and -13, as well as substrates for granzyme B (Fluorogenic substrates for caspase activity - Table 15.5). Granzyme B, a serine protease contained within cytotoxic T lymphocytes and natural killer cells, is thought to induce apoptosis in target cells by activating caspases and causing mitochondrial cytochrome c release.

EnzChek Caspase-3 Assay Kits

Molecular Probes' EnzChek Caspase-3 Assay Kits permit the detection of apoptosis by assaying for increases in caspase-3 and caspase-3–like protease activities (Figure 10.53, Figure 15.98). Our EnzChek Caspase-3 Assay Kit #1 (E13183) contains the 7-amino-4-methylcoumarin (AMC)–derived substrate Z-DEVD-AMC () (where Z represents a benzyloxycarbonyl group). This substrate, which is weakly fluorescent in the UV spectral range (excitation/emission maxima ~330/390 nm), yields the blue–fluorescent product AMC (A191, Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1, ), which has excitation/emission maxima of 342/441 nm upon proteolytic cleavage. The EnzChek Caspase-3 Assay Kit #2 (E13184) contains the R110-derived substrate, Z-DEVD-R110 (). This substrate is a bisamide derivative of R110, containing DEVD peptides covalently linked to each of R110's amino groups, thereby suppressing both the dye's visible absorption and fluorescence. Upon enzymatic cleavage by caspase-3 (or a closely related protease), the nonfluorescent bisamide substrate is converted in a two-step process first to the fluorescent monoamide and then to the even more fluorescent R110 (R6479, Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1, , Figure 10.52). Both of these hydrolysis products exhibit spectral properties similar to those of fluorescein, with excitation/emission maxima of 496/520 nm. The Z-DEVD-R110 substrate (R22120) is also available separately in a 20 mg unit size for high-throughput screening applications.

Either kit can be used to continuously measure the activity of caspase-3 and closely related proteases in cell extracts and purified enzyme preparations using a fluorescence microplate reader or fluorometer. AMC-based DEVD substrates, which yield blue fluorescence upon proteolytic cleavage, are widely used to monitor caspase-3 activity. The longer-wavelength spectra and higher extinction coefficient of the green-fluorescent products of the R110-based substrate in Kit #2 (E13184) should provide even greater sensitivity. The reversible aldehyde-based inhibitor Ac-DEVD-CHO can be used to confirm that the observed fluorescence signal in both induced and control cell populations is due to the activity of caspase-3–like proteases. Each kit contains:

• Z-DEVD-AMC (in Kit #1, E13183) or Z-DEVD-R110 (in Kit #2, E13184)
• Dimethylsulfoxide (DMSO)
• Concentrated cell-lysis buffer
• Concentrated reaction buffer
• Dithiothreitol (DTT)
• Ac-DEVD-CHO, a reversible aldehyde-based inhibitor
• 7-Amino-4-methylcoumarin (AMC) (in Kit E13183) or rhodamine 110 (in Kit E13184) reference standard to quantitate the amount of fluorophore released in the reaction
• Detailed protocols (EnzChek(R) Caspase-3 Assay Kit #1 *Z-DEVD-AMC Substrate*, EnzChek(R) Caspase-3 Assay Kit #2 *Z-DEVD-R110 Substrate*)

Each kit provides sufficient reagents for performing ~500 assays using a volume of 100 µL per assay.

RediPlate 96 EnzChek Caspase-3 Assay Kit

Our EnzChek Caspase-3 Assay Kit #2 is also available as a convenient RediPlate 96 EnzChek Caspase-3 Assay Kit (R35100, RediPlate 96 EnzChek(R) Caspase-3 Assay Kit), which includes one 96-well microplate, contained in a resealable foil packet to ensure the integrity of the fluorogenic components, plus all necessary buffers and reagents for performing the assay (Figure 10.54). The enzyme sample to be assayed is added to the microplate in a suitable buffer, along with any compounds to be tested. Then, after incubation, the resultant fluorescence is quantitated on a fluorescence microplate reader equipped with filters appropriate for the green-fluorescent R110, with excitation/emission maxima of 496/520 nm. The microplate consists of twelve removable strips, each with eight wells, allowing researchers to perform only as many assay as required for the experiment (Figure 8.60). Eleven of the strips (88 wells) are preloaded with the Z-DEVD-R110 substrate. The remaining strip, marked with blackened tabs, contains a dilution series of free R110 that may be used as a fluorescence reference standard. The reversible aldehyde-based inhibitor Ac-DEVD-CHO, which is supplied in a separate vial, can be used to confirm that the observed fluorescence signal in both induced and control cell populations is due to the activity of caspase-3–like proteases. RediPlate Assay Kits - Table 10.3 summarizes our other RediPlate 96 and RediPlate 384 Assay Kits for protease activity (Detecting Peptidases and Proteases - Section 10.4), phosphatase activity (Detecting Enzymes That Metabolize Phosphates and Polyphosphates - Section 10.3) and dsDNA quantitation (Nucleic Acid Detection and Quantitation in Solution - Section 8.3). Significant discounts apply to purchases of multiple units of all of our RediPlate products.

Image-iT LIVE Green Caspase Detection Kits for Fluorescence Microscopy

The Image-iT LIVE Green Caspase-3 and -7 Detection Kit, Image-iT LIVE Green Caspase-8 Detection Kit and Image-iT LIVE Green Poly Caspases Detection Kit (I35106, I35105, I35104) employ a novel approach to detect active caspases that is based on a fluorescent inhibitor of caspases (FLICA methodology). The FLICA inhibitor comprises a fluoromethyl ketone (FMK) moiety, which can react covalently with a cysteine, a caspase-selective amino acid sequence and a fluorescent carboxyfluorescein (FAM) reporter group. Essentially an affinity label, the FLICA inhibitor is thought to interact with the enzymatic reactive center of an activated caspase via the recognition sequence, and then to attach covalently to a cysteine through the reactive FMK moiety. The FLICA inhibitor's recognition sequence is aspartic acid–glutamic acid–valine–aspartic acid (DEVD) for caspase-3 and-7 detection, leucine–glutamic acid–threonine–aspartic acid (LETD) for caspase-8 detection and valine–alanine–aspartic acid (VAD) for detection of most caspases (including caspase-1, -3, -4, -5, -6, -7, -8 and -9). Importantly, the FLICA inhibitor is cell permeant and not cytotoxic; unbound FLICA molecules diffuse out of the cell and are washed away. The remaining green-fluorescent signal (excitation/emission maxima ~488/530 nm) can be used as a direct measure of the amount of active caspase that was present at the time the inhibitor was added. FLICA reagents have been used widely to study apoptosis with flow cytometry and microscopy. Recent work indicates that cellular fluorescence from the bound FLICA reagent is strongly linked to caspase activity in apoptotic cells; however, the interaction of the FLICA reagent with other cellular sites may contribute to signal intensity in nonapoptotic cells. Appropriate controls should be included in any experimental design.

Each Image-iT LIVE Green Caspase Detection Kit includes:

• FAM-DEVD-FMK caspase-3 and -7 reagent (in Kit I35106), FAM-LETD-FMK caspase-8 reagent (in Kit I35105) or FAM-VAD-FMK poly caspases reagent (in Kit I35104)
• Hoechst 33342
• Propidium iodide
• Dimethylsulfoxide (DMSO)
• Apoptosis fixative solution
• Concentrated apoptosis wash buffer
• Detailed protocols for fluorescence microcscopy assays (Image-iT(R) LIVE Green Caspase Detection Kits)

In addition to a specific FLICA reagent, each kit provides Hoechst 33342 and propidium iodide stains, which allow the simultaneous evaluation of caspase activation, nuclear morphology and plasma membrane integrity. Sufficient reagents are provided for 25 assays, based on labeling volumes of 300 µL. These Image-iT LIVE Green Caspase Detection Kits can also be used in combination with other reagents for multiparametric study of apoptosis.

Image-iT LIVE Red Caspase Detection Kits for Fluorescence Microscopy

The Image-iT LIVE Red Caspase-3 and -7 Detection Kit and Image-iT LIVE Red Poly Caspases Detection Kit (I35102, I35101) are analogous to the Image-iT LIVE Green Caspase Detection Kits except that the FLICA reagent contains a red-fluorescent sulforhodamine (SR) reporter group instead of a green-fluorescent carboxyfluorescein (FAM) reporter group. This assay's red-fluorescent signal (excitation/emission maxima ~550/595 nm) can be used as a direct measure of the amount of active caspase that was present at the time the inhibitor was added. Each Image-iT LIVE Red Caspase Detection Kit includes:

• SR-DEVD-FMK caspase-3 and -7 reagent (in Kit I35102) or SR-VAD-FMK poly caspases reagent (in Kit I35101)
• Hoechst 33342
• SYTOX Green nucleic acid stain
• Dimethylsulfoxide (DMSO)
• Apoptosis fixative solution
• Concentrated apoptosis wash buffer
• Detailed protocols for fluorescence microcscopy assays (Image-iT(R) LIVE Red Caspase Detection Kits)

In addition to a specific FLICA reagent, each kit provides Hoechst 33342 and SYTOX Green nucleic acid stains, which allow the simultaneous evaluation of caspase activation, nuclear morphology and plasma membrane integrity. Sufficient reagents are provided for 25 assays, based on labeling volumes of 300 µL.

Vybrant FAM Caspase Assay Kits for Flow Cytometry

Like the Image-iT Kits described above, the Vybrant FAM Caspase Assay Kits for flow cytometry are based on a fluorescent caspase inhibitor (FLICA methodology). We offer three different Vybrant FAM Caspase Assay Kits designed to target different caspases. The Vybrant FAM Caspase-3 and -7 Assay Kit (V35118) provides a FLICA inhibitor containing the caspase-3 and -7 recognition sequence DEVD; the Vybrant FAM Caspase-8 Assay Kit (V35119) provides a FLICA inhibitor containing the caspase-8 recognition sequence Leu-Glu-Thr-Asp (LETD); and the Vybrant FAM Poly Caspases Assay Kit (V35117) provides a FLICA inhibitor containing the caspase recognition sequence Val-Ala-Asp (VAD), which is recognized by caspase-1, -3, -4, -5, -6, -7, -8 and -9. In addition to the selective FLICA reagent, these kits contain the Hoechst 33342 and propidium iodide nucleic acid stains to permit simultaneous evaluation of caspase activation, membrane permeability and cell cycle. Each Vybrant FAM Caspase Assay Kit includes:

• FAM-DEVD-FMK caspase-3 and -7 reagent (in Kit V35118), FAM-LETD-FMK caspase-8 reagent (in Kit V35119) or FAM-VAD-FMK poly caspases reagent (in Kit V35117)
• Hoechst 33342
• Propidium iodide
• Dimethylsulfoxide (DMSO)
• Apoptosis fixative solution
• Concentrated apoptosis wash buffer
• Detailed protocols for flow cytometry assays (Vybrant(R) FAM Caspase-3 and -7 Assay Kit, Vybrant(R) FAM Caspase-8 Assay Kit, Vybrant(R) FAM Poly Caspases Assay Kit)

Sufficient reagents are provided for 25 assays, based on labeling volumes of 300 µL. These Vybrant FAM Caspase Assay Kits can be used in combination with other fluorescent probes, such as the far-red–fluorescent allophycocyanin annexin V (A35110), for a multiparameter study of apoptosis.

Cathepsins and Calpains

The role of intracellular cathepsins and calpains in apoptosis is unclear, although an upstream role of cathepsin B in activation of some caspases and cathepsins during apoptosis has been established. Pepstatin A, which is a selective inhibitor of carboxyl (acid) proteases such as cathepsin D, has been reported to inhibit apoptosis in microglia, lymphoid cells and HeLa cells. Consequently, our cell-permeant BODIPY FL pepstatin derivative (P12271), which we have shown to inhibit cathepsin D in vitro (IC₅₀ ~10 nM) and to target cathepsin D within lysosomes of live and fixed cells, may be of some utility in following the translocation of cathepsin D that may occur during apoptotic events.

Calpains are a family of ubiquitous calcium-activated thiol proteases that are implicated in a variety of cellular functions including exocytosis, cell fusion, apoptosis and cell proliferation. Caspase-dependent downstream processing of calpain has been reported, suggesting that calpain may play a role in the degradation phase of apoptosis that is distinct from that of caspases. One mechanism of caspase dependence appears to be processing of the endogenous calpain inhibitor calpastin by caspases. However, calpain activation has also been reported to be upstream of caspases in radiation-induced apoptosis. Our t-BOC-Leu-Met-CMAC fluorogenic substrate (A6520) has been used to measure calpain activity in hepatocytes following the addition of extracellular ATP and may be of utility in detecting caspase-activated processing of procalpain in live single cells. Peptidase substrates based on our CMAC fluorophore (7-amino-4-chloromethylcoumarin, C2110; Introduction to Enzyme Substrates and Their Reference Standards - Section 10.1) passively diffuse into several types of cells, where the thiol-reactive chloromethyl group is enzymatically conjugated to glutathione by intracellular glutathione S-transferase or reacts with protein thiols, thus transforming the substrate into a membrane-impermeant probe. Subsequent peptidase cleavage results in a bright blue-fluorescent glutathione conjugate; see Detecting Peptidases and Proteases - Section 10.4 for more information on AMC- and CMAC-based peptidase substrates.

A distinctive feature of the early stages of programmed cell death is the disruption of active mitochondria. This mitochondrial disruption includes changes in the membrane potential and alterations to the oxidation–reduction potential of the mitochondria. Changes in the membrane potential are presumed to be due to the opening of the mitochondrial permeability transition pore, allowing passage of ions and small molecules. The resulting equilibration of ions leads in turn to the decoupling of the respiratory chain and then the release of cytochrome c into the cytosol.

Mitochondrial Stains for Detecting Apoptosis

Molecular Probes has available several unique reagents for studying changes in the mitochondria during apoptosis.

• The green-fluorescent dye JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide, T3168; ) exists as a monomer at low concentrations or at low membrane potential. However, at higher concentrations — aqueous solutions above 0.1 µM — or at higher membrane potentials, JC-1 forms red-fluorescent "J-aggregates" (, , ) that exhibit a broad excitation spectrum and an emission maximum at ~590 nm (). Thus, the emission of this cyanine dye has been widely used to follow the changes in mitochondrial membrane potential that occur as a result of apoptosis (MitoProbe JC-1 Assay Kit, M34152; see below). JC-1 has been used successfully to follow mitochondrial dysfunction in apoptotic hippocampal neurons and opening of the mitochondrial permeability transition pore (MTP).
• Our JC-9 dye (3,3'-dimethyl-α-naphthoxacarbocyanine iodide, D22421, ) undergoes a similar potential-dependent spectral shift from a green-fluorescent product to a red-fluorescent aggregate () and is likely to be similarly useful for detecting apoptotic cells by both imaging and flow cytometry. Unlike JC-1, the green fluorescence of JC-9 is essentially invariant with membrane potential while the red fluorescence is significantly increased at hyperpolarized membrane potentials.
• MitoTracker Red CMXRos (M7512, ) provides quick, easy and reliable detection of the loss of mitochondrial membrane potential that occurs during apoptosis. Our Patented MitoTracker Red CMXRos probe can be fixed using aldehyde-based fixatives and can thus be detected through subsequent immunocytochemistry, DNA end-labeling, in situ hybridization or counterstaining steps. The changes in mitochondrial membrane potential in osteosarcoma cells observed using MitoTracker Red CMXRos were instrumental in demonstrating the ability of these cells to undergo reversible apoptosis without entering cell death. Ratiometric measurements that compare the fluorescence of the membrane potential–dependent MitoTracker Red CMXRos label to that of the membrane potential–independent MitoTracker Green FM dye (M7514, Probes for Mitochondria - Section 12.2) result in improved discrimination of apoptotic and nonapoptotic cell populations.
• Rhodamine 123 (R302; FluoroPure Grade - Note 19.2, R22420) is a cell-permeant, cationic, fluorescent dye that is readily sequestered by active mitochondria without inducing cytotoxic effects. Uptake and equilibration of rhodamine 123 is rapid — a few minutes — as compared with other membrane potential–sensitive dyes, which may take 30 minutes or longer. Although not aldehyde-fixable, rhodamine 123 allows for quick and easy detection of apoptotic cells.
• Most carbocyanine dyes with short (C₁–C₆) alkyl chains stain mitochondria of live cells when used at low concentrations (~0.5 µM or ~0.1 µg/mL) (MitoProbe DiIC₁(5) and DiOC₂(3) Assay Kits; M34151, M34150; see below). DiOC₆(3) (D273) is a green-fluorescent cationic dye that accumulates in active mitochondria and is useful in following changes in the membrane potential of the mitochondria that occur during programmed cell death. This dye has been used in flow cytometric analysis to study mitochondrial changes in apoptotic human myeloid leukemia cells.
• The accumulation of both the methyl and ethyl esters of tetramethylrhodamine (TMRM, T668; TMRE, T669) in mitochondria and endoplasmic reticulum is driven by membrane potential. TMRM has been used to study the temporal relationship between cytochrome c release from mitochondria and reduced mitochondrial membrane potential in apoptotic pheochromocytoma-6 cells and to investigate the mitochondrial permeability transition pore.
• Calcein, a green-fluorescent dye that is formed inside cells that are loaded using calcein AM (C1430, C3099, C3100MP; Viability and Cytotoxicity Assay Reagents - Section 15.2, ), can be taken up into the matrix of mitochondria due to opening of the mitochondrial permeability transition pore (MTP). The MTP allows relatively large molecules (less than 620 daltons) to pass from the cytosol into the mitochondrial matrix. The transport of calcein through the MTP has been used to study the role of the MTP in apoptosis.
• Nonyl acridine orange (A1372) is reported to bind to cardiolipin in the inner mitochondrial membrane. Its fluorescence decreases as cardiolipin becomes oxidized or otherwise altered during apoptosis.
• Our SYTO 16 green-fluorescent nucleic acid stain (S7578) shows decreased fluorescence in apoptotic cells that may be due to changes in mitochondrial DNA conformation. It is optimally excited by the 488 nm spectral line of the argon-ion laser, making it useful for both flow cytometry and confocal laser-scanning microscopy.

SelectFX Alexa Fluor 488 Cytochrome c Apoptosis Detection Kit

The SelectFX Alexa Fluor 488 Cytochrome c Apoptosis Detection Kit (S35115) provides all the reagents required to detect cytochrome c in fixed cells. Because cytochrome c is released from mitochondria into the cytosol during apoptosis, its localization can serve as a marker of apoptosis. This kit employs an anti–cytochrome c primary antibody and an Alexa Fluor 488 dye–labeled secondary antibody. The Alexa Fluor 488 dye exhibits bright green fluorescence that is compatible with filters and instrument settings appropriate for fluorescein. Each kit contains:

• Mouse IgG₁ anti–cytochrome c antibody
• Highly cross-adsorbed Alexa Fluor 488 goat anti–mouse IgG antibody
• Concentrated fixative solution
• Concentrated phosphate-buffered saline (PBS)
• Concentrated permeabilization solution
• Concentrated blocking solution
• Detailed protocols for mammalian cell preparation and staining (SelectFX(R) Alexa Fluor(R) 488 Cytochrome c Apoptosis Detection Kit)

The SelectFX Alexa Fluor 488 Cytochrome c Apoptosis Detection Kit can be used in conjunction with probes for other cell targets to achieve multicolor cell staining.

Image-iT LIVE Mitochondrial Transition Pore Assay Kit for Fluorescence Microscopy

The mitochondrial permeability transition pore, a nonspecific channel formed by components from the inner and outer mitochondrial membranes, appears to be involved in the release of mitochondrial components during apoptotic and necrotic cell death. In a healthy cell, the inner mitochondrial membrane is responsible for maintaining the electrochemical gradient that is essential for respiration and energy production. As Ca²⁺ is taken up and released by mitochondria, a low-conductance permeability transition pore appears to flicker between open and closed states. During cell death, the opening of the mitochondrial permeability transition pore dramatically alters the permeability of mitochondria. Continuous pore activation results from mitochondrial Ca²⁺ overload, oxidation of mitochondrial glutathione, increased levels of reactive oxygen species in mitochondria and other pro-apoptotic conditions. Cytochrome c release from mitochondria and loss of mitochondrial membrane potential are observed subsequent to continuous pore activation.

The Image-iT LIVE Mitochondrial Transition Pore Assay Kit (I35103), based on published experimentation for mitochondrial transition pore opening, provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone. This assay employs the acetoxymethyl (AM) ester of calcein, a colorless and nonfluorescent esterase substrate, and CoCl₂, a quencher of calcein fluorescence, to selectively label mitochondria. Cells are loaded with calcein AM, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. Once inside cells, calcein AM is cleaved by intracellular esterases to liberate the very polar fluorescent dye calcein, which does not cross the mitochondrial or plasma membranes in appreciable amounts over relatively short periods of time. The fluorescence from cytosolic calcein is quenched by the addition of CoCl₂, while the fluorescence from the mitochondrial calcein is maintained. As a control, cells that have been loaded with calcein AM and CoCl₂ can also be treated with a Ca²⁺ ionophore such as ionomycin (I24222, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents - Section 19.8) to allow entry of excess Ca²⁺ into the cells, which triggers mitochondrial pore activation and subsequent loss of mitochondrial calcein fluorescence. This ionomycin response can be blocked with cyclosporine A, a compound reported to prevent mitochondrial transition pore formation by binding cyclophilin D.

The Image-iT LIVE Mitochondrial Transition Pore Assay Kit has been tested with HeLa cells and bovine pulmonary artery endothelial cells (BPAEC). Each Image-iT LIVE Mitochondrial Transition Pore Assay Kit provides:

• Calcein AM
• MitoTracker Red CMXRos, a red-fluorescent mitochondrial stain (excitation/emission maxima ~579/599 nm)
• Hoechst 33342, a blue-fluorescent nuclear stain (excitation/emission maxima ~350/461 nm)
• Ionomycin
• CoCl₂
• Dimethylsulfoxide (DMSO)
• A detailed protocol (Image-iT(R) LIVE Mitochondrial Transition Pore Assay Kit)

Sufficient reagents are provided for 100 assays, based on labeling volumes of 1 mL.

MitoProbe Transition Pore Assay Kit for Flow Cytometry

The MitoProbe Transition Pore Assay Kit (M34153), based on published experimentation for mitochondrial transition pore opening, provides a more direct method of measuring mitochondrial permeability transition pore opening than assays relying on mitochondrial membrane potential alone (Figure 15.101). As with the Image-iT LIVE mitochondrial transition pore assay described above, this assay employs the acetoxymethyl (AM) ester of calcein, a colorless and nonfluorescent esterase substrate, and CoCl₂, a quencher of calcein fluorescence, to selectively label mitochondria. Cells are loaded with calcein AM, which passively diffuses into the cells and accumulates in cytosolic compartments, including the mitochondria. Once inside cells, calcein AM is cleaved by intracellular esterases to liberate the very polar fluorescent dye calcein, which does not cross the mitochondrial or plasma membranes in appreciable amounts over relatively short periods of time. The fluorescence from cytosolic calcein is quenched by the addition of CoCl₂, while the fluorescence from the mitochondrial calcein is maintained. As a control, cells that have been loaded with calcein AM and CoCl₂ can also be treated with a Ca²⁺ ionophore such as ionomycin (I24222, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents - Section 19.8) to allow entry of excess Ca²⁺ into the cells, which triggers mitochondrial pore activation and subsequent loss of mitochondrial calcein fluorescence. This ionomycin response can be blocked with cyclosporine A, a compound reported to prevent mitochondrial transition pore formation by binding cyclophilin D.

The MitoProbe Transition Pore Assay Kit has been tested with Jurkat cells, MH1C1 cells and bovine pulmonary artery endothelial cells (BPAEC). Each MitoProbe Transition Pore Assay Kit provides:

• Calcein AM
• CoCl₂
• Ionomycin
• Dimethylsulfoxide (DMSO)
• A detailed protocol (MitoProbe Transition Pore Assay Kit)

Sufficient reagents are provided for 100 assays, based on labeling volumes of 1 mL.

MitoProbe JC-1 Assay Kit for Flow Cytometry

The MitoProbe JC-1 Assay Kit (M34152) provides the cationic dye JC-1 and a mitochondrial membrane potential disrupter, CCCP (carbonyl cyanide 3-chlorophenylhydrazone), for the study of mitochondrial membrane potential. JC-1 () exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (~529 nm) to red (~590 nm), due to concentration-dependent formation of red-fluorescent J-aggregates. Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio, which is dependent only on the membrane potential and not on other factors such as mitochondrial size, shape and density, which may influence single-component fluorescence measurements. Use of fluorescence ratio detection therefore allows researchers to make comparative measurements of membrane potential and to determine the percentage of mitochondria within a population that respond to an applied stimulus. Subtle heterogeneity in cellular responses can be discerned in this way. For example, four distinct patterns of mitochondrial membrane potential change in response to glutamate receptor activation in neurons have been identified using confocal ratio imaging of JC-1 fluorescence.

JC-1 can be used as an indicator of mitochondrial potential in a variety of cell types, including myocytes and neurons, as well as in intact tissues and isolated mitochondria. JC-1 is more specific for mitochondrial versus plasma membrane potential and more consistent in its response to depolarization than some other cationic dyes such as DiOC₆(3) and rhodamine 123. The most widely implemented application for JC-1 is the detection of mitochondrial depolarization occurring in apoptosis (Figure 15.102). Each MitoProbe JC-1 Assay Kit provides:

• JC-1
• Dimethylsulfoxide (DMSO)
• CCCP
• Concentrated phosphate-buffered saline (PBS)
• A detailed protocol (MitoProbe JC-1 Assay Kit for Flow Cytometry)

Sufficient reagents are provided for 100 assays, based on a labeling volume of 1 mL.

MitoProbe DiIC₁(5) and MitoProbe DiOC₂(3) Assay Kits for Flow Cytometry

Cationic carbocyanine dyes have been shown to accumulate in cells in response to membrane potential, and membrane potential changes have been studied in association with apoptosis. The MitoProbe DiIC₁(5) and MitoProbe DiOC₂(3) Assay Kits (M34151, M34150) provide solutions of the far-red–fluorescent DiIC₁(5) (1,1',3,3,3',3'-hexamethylindodicarbocyanine iodide) and green-fluorescent DiOC₂(3) (3,3'-diethyloxacarbocyanine iodide) carbocyanine dyes, respectively, along with a mitochondrial membrane potential disrupter, CCCP, for the study of mitochondrial membrane potential. These DiIC₁(5) and DiOC₂(3) carbocyanine dyes penetrate the cytosol of eukaryotic cells and, at concentrations below 100 nM, accumulate primarily in mitochondria with active membrane potentials. In the case of DiOC₂(3), this accumulation is accompanied by a shift from green to red emission due to dye stacking (Figure 22.17), allowing the use of a ratiometric parameter (red/green fluorescence ratio) that corrects for size differences when measuring membrane potential in bacteria. DiIC₁(5) and DiOC₂(3) stain intensities decrease when cells are treated with reagents that disrupt mitochondrial membrane potential, such as CCCP. Each MitoProbe DiIC₁(5) and MitoProbe DiOC₂(3) Assay Kit provides:

• DiIC₁(5) (in Kit M34151) or DiOC₂(3) (in Kit M34150)
• CCCP
• Detailed protocols for labeling cells with the short-chain carbocyanine dye, as well as with annexin V conjugates (not included) (MitoProbe DiIC{1}(5) Assay Kit for Flow Cytometry, MitoProbe DiOC{2}(3) Assay Kit for Flow Cytometry)

Cells stained with DiIC₁(5) can be visualized by flow cytometry with red excitation and far-red emission filters; cells stained with DiOC₂(3) can be visualized by flow cytometry with blue excitation and green and red emission filters. DiIC₁(5) can be paired with other reagents, such as propidium iodide and the green-fluorescent Alexa Fluor 488 annexin V (both provided in the Vybrant Apoptosis Assay Kit #2, V13241), for multiparameter study of vitality and apoptosis. DiOC₂(3) can be paired with other reagents, such as the far-red–fluorescent allophycocyanin annexin V (A35110), for multiparameter study of vitality and apoptosis (Figure 15.103). Combining these short-chain carbocyanine dyes with annexin V conjugates results in superior resolution of subpopulations when compared with results obtained with other commonly used dyes.

The bcl-2 proto-oncogene product is reported to play a role in preventing apoptosis through its antioxidant properties. Following an apoptotic signal, cells sustain progressive lipid peroxidation — as detected with cis-parinaric acid (P36005) — that can be suppressed by bcl-2 overexpression. cis-Parinaric acid was also used to assess lipid peroxidation in Down syndrome neurons, which exhibit increased levels of intracellular reactive oxygen species that lead to a reduction in levels of intracellular reduced glutathione and apoptosis. The reagent diphenyl-1-pyrenylphosphine (DPPP, D7894) is essentially nonfluorescent until it is oxidized by hydroperoxides to a phosphine oxide. Its lipid solubility makes DPPP similarly useful for detecting hydroperoxides in the membranes of live cells.

Induction of apoptosis in human natural killer (NK) cells by monocytes is blocked by catalase, a scavenger of hydrogen peroxide, and by sodium azide, a myeloperoxidase inhibitor, whereas scavengers of superoxide and hydroxyl radicals do not prevent apoptosis. The most common fluorogenic probe for detecting reactive oxygen species is 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA, D399), which has been used to examine the effect of caspase-3 inhibitors on hydrogen peroxide production during apoptosis, in apoptotic embryos and in chemosensitive or chemoresistant cancer cells. H₂DCFDA can detect so-called "necrotic zones" containing cells under oxidative stress in tissues; however, for this application we recommend our 5-(and 6-)chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA, C6827; ), which has greater cell-membrane permeability and better cell retention of its green-fluorescent oxidation product. The acetoxymethyl ester of 2',7'-dichlorodihydrofluorescein diacetate (C2938, ) is also more permeant to live cells and tissues and has been used to detect hydrogen peroxide in transplanted myoblasts. As would be expected, the other major probes for reactive oxygen species — dihydrorhodamine 123 (D632, D23806; ) and dihydroethidium (hydroethidine; D1168, D11347, D23107), each of which is colorless and nonfluorescent until oxidized to the mitochondrial probe rhodamine 123 or to the nucleic acid stain ethidium — are also useful for detecting apoptotic cells in culture, and likely in tissues. Another scavenger of reactive oxygen species, dihydrocalcein AM (D23805, ) yields the green-fluorescent dye calcein () upon intracellular oxidation; the cell retention of calcein is superior to that of most other dyes (Figure 15.3). The principal oxidant of these probes is reportedly peroxynitrite, which is generated from nitric oxide (Probes for Nitric Oxide Research - Section 18.3), although superoxide has also been implicated.

Probes such as 10-acetyl-3,7-dihydroxyphenoxazine (the Amplex Red reagent, A12222, A22177; Generating and Detecting Reactive Oxygen Species - Section 18.2) and the Amplex UltraRed reagent (A36006) react with hydrogen peroxide in the presence of a peroxidase to form red-fluorescent resorufin derivatives and may therefore be useful for correlating hydrogen peroxide production in cells with apoptosis. All of our probes for detecting reactive oxygen species are described in Probes for Reactive Oxygen Species, Including Nitric Oxide - Chapter 18.