15.5 Assays for Apoptosis

Transcription

1 15.5 Assays for Apoptosis Apoptosis (programmed cell death) is the genetically controlled ablation of cells during development. 1 Stroke-damaged neurons and cells experiencing deregulation of the cell cycle, such as tumor cells or those subjected to genetic transformation, are also prone to apoptosis. 2 4 Apoptosis is distinct from necrosis in both the biochemical and the morphological changes that occur. 5 9 In contrast to necrotic cells, apoptotic cells are characterized morphologically by compaction of the nuclear chromatin, shrinkage of the cytoplasm and production of membrane-bound apoptotic bodies. Biochemically, apoptosis is distinguished by fragmentation of the genome and cleavage or degradation of several cellular proteins. As with cell viability, no single parameter fully defines cell death; therefore, it is often advantageous to use several different approaches when studying apoptosis. Several methods have been developed to distinguish live cells from early and late apoptotic cells and from necrotic cells; these are described below and in a number of review articles and seminal publications. 6,10 16 Anticancer drug candidates failing to induce apoptosis are likely to have decreased clinical efficacy, 17 making apoptosis assays important tools for high-throughput drug screening. Apoptotic cells are typically eliminated by phagocytosis; thus, apoptotic cells that have been selectively labeled with a fluorescent dye can potentially be used as tracers for phagocytosis, 18 a cell process that is discussed in Section Apoptosis Assays Using Nucleic Acid Stains 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 Section 8.1, have been used to detect apoptotic cells by fluorescence imaging or flow cytometry. 19 Our YO-PRO-1 (Y-3603) nucleic acid stain is the basis of an important assay for apoptotic cells that is compatible with both fluorescence microscopy and flow cytometry. 20 Selective uptake of YO-PRO-1 by apoptotic cells of a dexamethasonetreated population of thymocytes, an irradiated peripheral blood mononuclear cell population and a growth factor depleted tumor B cell line was confirmed by cell sorting. 21 Unlike Hoechst 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 (Figure 15.65) 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 (V-13243, V-23201, 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 (S-7575, S-7578), are proving useful for distinguishing apoptotic neuronal cells 22 and apoptotic thymocytes. 23 Our SYTO Fluorescent Nucleic Acid Stain Sampler Kits (S-7554, S-7572, S-11340, S-11350, S-11360; Section 8.1) provide fluorescent SYTO dyes covering the entire visible spectrum (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 (S-7579) than with either fluoresceinlabeled annexin V or propidium iodide. 24 Hoechst (H-1399, H-3570; FluoroPure grade, H-21492) is readily taken up by cells during the initial stages of apoptosis, whereas cell-impermeant dyes such as propidium iodide (P-1304, P-3566, P-21493; Section 8.1) and ethidium bromide (E-1305, E-3565; 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 and propidium iodide has been extensively used for simultaneous flow cytometric and fluorescence imaging analysis of the stages of apoptosis and cell-cycle distribution. 25,26,28 30 Our Vybrant Apoptosis Assay Kit #5 (V-13244, see below) is based on these reagents and our Vybrant Apoptosis Assay Kit #7 (V-23201, 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 uptake in partially apoptotic cell populations is correlated with low intracellular ph, as measured with our carboxy SNARF-1 ph indicator 31 (C-1271, C-1272; Section 21.2). Hoechst 33342, which selectively stains nuclei of apoptotic cells blue fluorescent, has also been used in combination with calcein AM (C-1430, C-3099, C-3100; Section 15.2), which labels all cells that have intact membranes even apoptotic cells green fluorescent. 32,33 Presumably the dead-cell population could be selectively detected using propidium iodide to make this a three-color assay. Figure Absorption and fluorescence emission spectra of YO-PRO-1 (Y-3603) bound to DNA. Section

2 7-Aminoactinomycin D (7-AAD, A-1310) has been used alone or in combination with Hoechst 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 AAD has also been used in combination with the green-fluorescent SYTO 16 nucleic acid stain (S-7578) to detect early stages of apoptosis that could not be detected by 7-AAD alone. 40 The cell-permeant nucleic acid stain LDS 751 (L-7595) has been used to discriminate intact nucleated cells from nonnucleated cells and cells with damaged nuclei, 41,42 as well as to differentiate apoptotic cells from nonapoptotic cells. 23,43 Acridine orange (A-1301, A-3568) exhibits metachromatic fluorescence that is sensitive to DNA conformation, making it a useful probe for detecting apoptotic cells. 44 When analyzed by flow cytometry, apoptotic cells stained by acridine orange show reduced green fluorescence and enhanced red fluorescence in comparison to normal cells. 45 DAPI (D-1306, D-21490; Section 8.1) and sulforhodamine 101 (S-359, Section 14.3) can be used together in fixed apoptotic cells to reveal concomitant breakdown of proteins and DNA. 6,45 47 The excited-state lifetime of ethidium homodimer-2 (E-3599, Section 8.1) has been shown to be different in populations of aldehyde-fixed apoptotic and nonapoptotic cells. 48 Ethidium monoazide (E-1374, 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. 49 Figure DNA extracts from camptothecintreated HL-60 cells separated on an agarose gel and stained with SYBR Green I nucleic acid gel stain (S-7563, S-7567, S-7585). The 200 to 5000 bp DNA fragments characteristic of apoptotic cells (which appear as ladders ) are clearly visualized with this sensitive nucleic acid stain. Cell preparations were gifts of Zbigniew Darzynkiewicz, Cancer Research Institute, New York Medical College. Table 15.4 Summary of Molecular Probes Vybrant Apoptosis Assay Kits. Cat # Kit Name Probe(s) for Apoptotic Cells (Abs/Em) * V Vybrant Apoptosis Assay Kit #1 V Vybrant Apoptosis Assay Kit #2 V Vybrant Apoptosis Assay Kit #3 V Vybrant Apoptosis Assay Kit #4 V Vybrant Apoptosis Assay Kit #5 V Vybrant Apoptosis Assay Kit #6 V Vybrant Apoptosis Assay Kit #7 Alexa Fluor 488 annexin V (495/519) Alexa Fluor 488 annexin V (495/519) FITC annexin V (494/519) YO-PRO-1 dye (491/509) Hoechst (346/480) Biotin-X annexin V and Alexa Fluor 350 streptavidin (345/442) Hoechst (346/480) and YO-PRO-1 dye (491/509) * Approximate absorption and emission maxima, in nm. Probe for Necrotic Cells (Abs/Em) * SYTOX Green nucleic acid stain (504/523) Propidium iodide (535/617) Propidium iodide (535/617) Propidium iodide (535/617) Propidium iodide (535/617) Propidium iodide (535/617) Propidium iodide (535/617) Number of Assays 50 flow cytometry assays, each containing to cells in a 1 ml volume 50 flow cytometry assays, each containing to cells in a 1 ml volume 50 flow cytometry assays, each containing to cells in a 1 ml volume 200 flow cytometry assays, each containing to cells in a 1 ml volume 200 flow cytometry assays, each containing to cells in a 1 ml volume 200 flow cytometry assays, each containing to cells in a 1 ml volume 200 flow cytometry assays, each containing to cells in a 1 ml volume Kit Features Because the Alexa Fluor 488 annexin V based assay in Kit #1 uses only the green fluorescence channel on the flow cytometer, other parameters can be measured simultaneously using fluorescent probes that have different emission spectra. In Kit #2, apoptotic cells are labeled with annexin V conjugated to our exceptionally bright and photostable green-fluorescent Alexa Fluor 488 dye. Necrotic cells are labeled with red-fluorescent propidium iodide. Kit #3 is identical to Kit #2 except that it contains the fluorescein conjugate of annexin V. Kit #4 detects changes in cell membrane permeability with YO-PRO-1 dye, a green-fluorescent nucleic acid stain that is permeant to apoptotic cells but not to live cells. Necrotic cells are labeled with red-fluorescent propidium iodide. Kit #5 uses Hoechst in combination with propidium iodide to distinguish between the condensed chromatin of apoptotic cells and the looser chromatin structure in live cells. Kit #6 is identical to Kit #2 except that it contains the biotin-x conjugate of annexin V, as well as Alexa Fluor 350 streptavidin for the secondary detection of annexin V binding. Kit #7 is a combination of Kits #4 and #5. All three dyes can be excited by a UV laser, or by a combination of UV and 488 nm excitation. 648 Chapter 15 Assays for Cell Viability, Proliferation and Function

3 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. 46,50 53 Ethidium bromide has been used for a dot-blot assay to detect apoptotic DNA fragments. 54 Our ultrasensitive SYBR Green I nucleic acid stain (S-7567, Section 8.4) and SYBR DX DNA blot stain (S-7550, Section 8.5) allow the detection of even fewer apoptotic cells in these applications (Figure 15.66). 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) (Figure 15.69). Vybrant Apoptosis Assay Kit #4 Our Vybrant Apoptosis Assay Kit #4 (V-13243) detects apoptosis on the basis of changes that occur in the permeability of cell membranes (Table 15.4). 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. 21,32,55 57 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 15.67) and fluorescence microscopy (Figure 15.68). 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 Table Vybrant Apoptosis Assay Kits #5 and #7 The Vybrant Apoptosis Assay Kit #5 (V-13244) 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 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 (Table 15.4). 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. 6,28,58 The 351 nm spectral line of an argon-ion laser or other suitable UV source is required for excitation of the Hoechst dye, whereas propidium iodide may 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 Table The Vybrant Apoptosis Assay Kit #7 combines the detection principles used in our Vybrant Apoptosis Assay Kits #4 (see above) and #5. Three nucleic acid stains Hoechst 33342, YO-PRO-1 and propidium iodide are utilized to identify by flow cytometry the fully live-cell population by their blue fluorescence, 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 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 Table Figure Jurkat human T-cell leukemia cells treated with 10 µm camptothecin for four hours (bottom panel) or untreated (as control, top panel). Cells were then treated with the reagents in the Vybrant Apoptosis Assay Kit #4 (V-13243) followed by flow cytometric analysis. 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, N = necrotic cells. Figure Apoptosis induced in Jurkat cells with 10 µm camptothecin. The cells were then treated with the reagents in the Vybrant Apoptosis Assay Kit #4 (V-13243). Viable cell nuclei were labeled with YO-PRO-1 dye (green) (Y-3603). Late-stage apoptotic and necrotic cells were detected with propidium iodide (red) (P-1304, P-3566, P-21493). Section

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. 59,60 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 (Figure 15.69). 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, 61 evaluate DNA replicative integrity, 62 monitor the sensitivity of tumor cells to radiation damage 63 and assess the sensitivity of molluscan cells to toxins in the environment. 64 The Comet assay can also be used in combination with FISH (Section 8.5) to identify specific sequences with damaged DNA. 63 Comet assays have traditionally been performed using ethidium bromide to stain the DNA. 59 However, our YOYO-1 dye was found to increase the sensitivity of the assay eightfold compared to ethidium bromide. 60 Use of the SYBR Gold and SYBR Green I stains 65 improves the sensitivity of this assay. Detecting DNA Strand Breaks with ChromaTide Nucleotides 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 frag- ments in a template-independent fashion. 19,66 69 Break sites have traditionally been labeled with ChromaTide biotin-11-dutp (C-11411), followed by subsequent detection with an avidin or streptavidin conjugate (Section 7.6, Table 7.17). 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 (C-7614) as a TdT substrate 74,75 (Figure 15.70). 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 74 (Figure 15.70). Moreover, it has been reported that 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 Texas Red-12-dUTP (C-7631) has been used similarly for a TdT-mediated apoptosis assay; 76 presumably a number of the ChromaTide dutp nucleotides listed in Table 8.6 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 (Section 7.4) can amplify the signal of many of the dyes used to prepare the ChromaTide nucleotides. 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 amyo- Figure Comet assay with SYBR Green I nucleic acid gel stain (S-7563, S-7567, S-7585). DNA fragmentation associated with oxidative DNA damage was visualized using Trevigen s CometAssay kit. HL-60 cells were treated with H 2 O 2 and immobilized onto a Trevigen CometSlide for analysis. The cells were gently lysed, washed and treated with endonuclease. Slides were subjected to electrophoresis in alkaline electrophoresis buffer and stained with SYBR Green I stain. Figure HL-60 cells treated with camptothecin for three hours. The DNA strand nicks characteristic of apoptosis were detected with the TUNEL (terminal deoxynucleotidyl transferase mediated dutp nick end-labeling) assay using the fluorescently labeled nucleotide, ChromaTide BODIPY FL-14-dUTP (C-7614). Image contributed by Zbigniew Darzynkiewicz, Cancer Research Institute, New York Medical College. 650 Chapter 15 Assays for Cell Viability, Proliferation and Function

5 trophic lateral sclerosis. 81 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, 82,83 which appear as a relatively early step in some apoptotic processes Because our ChromaTide BODIPY FL-14-dUTP (C-7614), ChromaTide fluorescein-12-dutp 87,88 (C-7604) and ChromaTide biotin-16 dutp (C-11411) 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. APO-BrdU TUNEL Assay Kit Because DNA fragmentation is one of the most reliable methods for detecting apoptosis, 84 we have collaborated with Phoenix Flow Systems to offer the APO-BrdU TUNEL Assay Kit (A-23210), 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 (Figure 15.71). 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: Figure Human lymphoma cells treated with camptothecin for four hours and stained using the APO-BrdU TUNEL Assay Kit (A-23210). Cells containing DNA strand nicks characteristic of apoptosis are detected by TUNEL and fluoresce green, while necrotic cells are stained with redfluorescent propidium iodide. 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 Sufficient reagents are provided for approximately 60 assays of 1 ml samples, each containing cells/ml. Apoptosis Assays Using Annexin V Conjugates Annexin V Conjugates Molecular Probes is collaborating with Nexins Research BV the original developer and patent holder 89 of fluorescent phosphatidylserine-binding proteins to provide what we feel are the best and brightest annexin V conjugates available. The human anticoagulant annexin V is a kilodalton, Ca 2+ -dependent phospholipid-binding protein that has a high affinity for phosphatidylserine (PS). 90,91 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, where it can be detected by annexin V conjugates. Highly fluorescent annexin V conjugates provide quick and reliable detection methods for studying the externalization of phosphatidylserine, 13 15,92,93 an indicator of intermediate stages of apoptosis. Nuclear fragmentation, mitochondrial membrane potential flux and caspase-3 activation apparently precede phosphatidylserine flipping during apoptosis, while 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 15.72). Annexin V conjugates 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 Figure Jurkat human T-cell leukemia cells treated with 10 µm camptothecin for four hours (bottom panel) or untreated (as control, top panel). Cells were then treated with the reagents in the Vybrant Apoptosis Assay Kit #2 (V-13241), followed by flow cytometric analysis. 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, N = necrotic cells. Section

6 of apoptotic and nonapoptotic cells. 94 Our annexin V conjugates are available as standalone reagents, each suitable for at least 100 flow cytometric assays or many more imaging assays, or in several variations of our Vybrant Apoptosis Assay Kits (Table 15.4). Our annexin V conjugates include: Figure Jurkat human T-cell leukemia cells treated with 1 µm camptothecin. The externalized phosphatidylserine, a characteristic of early-stage apoptotic cells, was detected with Alexa Fluor 488 annexin V (A-13201). The late-stage apoptotic and necrotic cells were stained with propidium iodide (P-1304, P-3566, P-21493). The image was acquired using bandpass filters appropriate for fluorescein. Figure Jurkat human T-cell leukemia cells treated with 10 µm camptothecin for four hours (black line) or untreated (as control, blue line). Cells were then treated with the reagents in the Vybrant Apoptosis Assay Kit #1 (V-13240), followed by flow cytometric analysis. 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). Our fluorogenic caspase substrates are available in bulk from Molecular Probes for high-throughput screening applications. Contact custom@probes.com for more information. Alexa Fluor 488 annexin V 95 (A-13201, Figure 15.73), 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 1.10, Figure 1.48). Alexa Fluor 488 annexin V is used in both our Vybrant Apoptosis Assays Kits #1 and #2 (V-13240, V-13241; 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 (A-13199), a green-fluorescent conjugate that has been extensively used by a number of laboratories to detect apoptotic cells populations. 14,15,24,92,94 Fluorescein annexin V is frequently used in combination with propidium iodide to detect necrotic cells, as in our Vybrant Apoptosis Assay Kit #3 (V-13242, see below). Oregon Green 488 annexin V (A-13200), a green-fluorescent conjugate that is spectrally similar to the fluorescein annexin V conjugate but is brighter and more photostable (Figure 1.42). Alexa Fluor 568 annexin V (A-13202), 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 (A-21335, Section 7.5), for multiparametric experiments. Alexa Fluor 594 annexin V 96,97 (A-13203), 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 (A-23204), which permits use of long-wavelength excitation sources for detection of apoptotic cells by either flow cytometry or microscopy. Alexa Fluor 350 annexin V (A-23202) can be excited in the ultraviolet and has brightblue fluorescence. Alternatively, the reagents in our Vybrant Apoptosis Assay Kit #6 (V-23200) can be used in this spectral region. Biotin-X annexin V 98,99 (A-13204), which can be detected by any of our fluorescent avidin or streptavidin conjugates (Section 7.6), gives the researcher the ultimate in color selection for multiparametric experiments. Biotin-X annexin V also permits detection of apoptotic cells by electron microscopy 100 and should permit separation of apoptotic cells with our Captivate ferrofluid streptavidin conjugate (C-21476, Section 7.6). Table 15.5 Fluorogenic substrates for caspase activity. Substrate * Enzymes Cat # Z-DEVD-AMC Caspase-3, Caspase-7 E Z-DEVD-R110 Caspase-3, Caspase-7 E R Z-DEVD-AFC Caspase-3, Caspase-7 A R110, bis-l-aspartic acid amide (D 2 -R110) Caspase-3, Caspase-7 R Z-IETD-AFC Caspase-8 A Z-IETD-AMC Caspase-8 A Z-IETD-R110 Caspase-8 R R * AMC = 7-amino-4-methylcoumarin; R110 = rhodamine 110; AFC = 7-amino-4-trifluoromethylcoumarin. The absorption and emission maxima for the cleaved fluorophores are: 342/441 nm for AMC, 376/499 nm for AFC and 496/520 nm for R Chapter 15 Assays for Cell Viability, Proliferation and Function

7 Vybrant Apoptosis Assay Kit #1 With the Vybrant Apoptosis Assay Kit #1 (V-13240), apoptotic cells are detected on the basis of the externalization of phosphatidylserine. This kit contains recombinant annexin V conjugated to the Alexa Fluor 488 dye, one of our brightest and most photostable green fluorophores 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 15.74). These populations can easily be distinguished using a flow cytometer with the 488 nm spectral line of an argonion laser for excitation. Both Alexa Fluor 488 annexin and the SYTOX Green dye emit a green fluorescence that can be detected in the FL1 channel, freeing the other channels for the detection of additional probes in multicolor labeling experiments. 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 Table Vybrant Apoptosis Assay Kit #2 Like the Vybrant Apoptosis Kit #1, our Vybrant Apoptosis Assay Kit #2 (V-13241) detects the externalization of phosphatidylserine in apoptotic cells (Table 15.4). 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 15.72). 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 Table Vybrant Apoptosis Assay Kit #3 The Vybrant Apoptosis Assay Kit #3 (V-13242) 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 (Table 15.4). The kit components, number of assays and assay principles are summarized in Table Vybrant Apoptosis Assay Kit #6 The Vybrant Apoptosis Assay Kit #6 (V-23200) 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 (Table 15.4). 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 (FL1) 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 Table Apoptosis Assays Based on Protease Activity Caspases Caspases comprise a key component of the apoptotic machinery of cells, participating in an enzyme cascade that results in cellular disassembly. 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. These proteases are typically synthesized as inactive precursors. Inhibitor release or cofactor binding activates the caspase through cleavage at internal aspartates through autocatalysis or by the action of another protease. 101 Caspase-3 Substrates and Assay Kits Caspase-3 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. 102,103 Molecular Probes offers a selection of fluorogenic substrates (Table 15.5) containing the caspase-3 recognition site Asp-Glu- Val-Asp (DEVD); in particular our EnzChek Caspase-3 Assay Kits #1 and #2 provide a simple and direct assay of caspase-3 (Figure 15.75) and other DEVD-specific protease activities (e.g., caspase-7). Each kit contains: Z-DEVD-AMC 104,105 (in Kit E-13183) or Z-DEVD- R (in Kit E-13184) Dimethylsulfoxide (DMSO) Concentrated cell-lysis buffer Concentrated reaction buffer Dithiothreitol (DTT) Ac-DEVD-CHO, a reversible aldehyde inhibitor 7-Amino-4-methylcoumarin (AMC) (in Kit E-13183) or rhodamine 110 (in Kit E-13184) reference standard to quantitate the amount of fluorophore released in the reaction Detailed protocols Our EnzChek Caspase-3 Assay Kit #1 (E-13183) contains the 7-amino-4-methylcoumarin (AMC) derived substrate Z-DEVD- AMC (Figure 15.76) (where Z represents a benzyloxycarbonyl Section

8 group). This substrate, which is weakly fluorescent in the UV spectral range (excitation/emission maxima ~330/390 nm), yields the blue fluorescent product AMC (A-191, Section 10.1, Figure 10.3), which has excitation/emission maxima of 342/441 nm upon proteolytic cleavage. The EnzChek Caspase-3 Assay Kit #2 (E-13184) contains the rhodamine 110 (R110) derived substrate, Z-DEVD-R (Figure 15.77). 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 (R-6479; Section 10.1; Figure 10.4, Figure 10.44). 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 (R-22120) 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. The reversible aldehyde inhibitor Ac-DEVD- CHO 103 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 of the kits contains sufficient reagents for about 500 assays using 100 µl volumes. Also for assaying caspase-3 activity we offer Z-DEVD- AFC 110,111 (A-22121), which undergoes an ~65 nm red-shift to exhibit a peak emission of ~499 nm upon cleavage) and the bis-laspartic acid amide of R110 (D 2 -R110, R-22122), which contains R110 flanked by aspartic acid residues (Table 15.5). D 2 -R110 does not appear to require any invasive techniques such as osmotic shock to gain entrance into the cytoplasm (Figure 15.78). It may serve as a substrate for a variety of apoptosis-related proteases, including caspase-3 and caspase 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. 112,113 The activated protease has been shown to be involved in a pathway that mediates the release of cytochrome c from the mitochondria 114 and is also known to activate downstream caspases, such as caspase Three fluorogenic substrates containing the caspase-8 recognition sequence Ile-Glu-Thr-Asp (IETD) are available (Table 15.5); Z-IETD-AMC and Z-IETD-AFC (A-22127, A-22128; blue fluorescent after cleavage) and Z-IETD-R (R-22125, R-22126; green fluorescent after cleavage). 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 116,117 and cathepsins during apoptosis has been established. 118 Pepstatin A (P-6542), 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 (P-12271), which we have shown to inhibit cathepsin D in vitro (IC 50 ~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. 122,123 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. 121,124 Caspase-dependent downstream processing of calpain Figure E Z-DEVD-AMC substrate included in the EnzChek Caspase-3 Assay Kit #1. Figure Detection of protease activity in Jurkat cells using the EnzChek Caspase-3 Assay Kit #1 with Z-DEVD-AMC substrate (E-13183). Cells were either treated with 10 µm camptothecin for four hours at 37 C to induce apoptosis (induced) or left untreated (control). Both induced and control cells were then harvested, lysed and assayed. Reactions were carried out at room temperature, and fluorescence was measured in a fluorescence microplate reader using excitation at 360 ± 20 nm with emission detection at 460 ± 20 nm after the indicated amount of time. Figure E Z-DEVD-R110 substrate included in the EnzChek Caspase-3 Assay Kit # Chapter 15 Assays for Cell Viability, Proliferation and Function

9 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 caspase(s). 128 However, calpain activation has also been reported to be upstream of caspases in radiation-induced apoptosis. 129 Our t-boc-leu-met-cmac fluorogenic substrate (A-6520) has been used to measure calpain activity in hepatocytes following the addition of extracellular ATP 130 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, C-2110; 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 (Section 10.4). Apoptosis Assays Using Mitochondrial Stains A distinctive feature of the early stages of programmed cell death is the disruption of active mitochondria This mitochondria 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. 134,135 Molecular Probes has available several unique reagents for studying changes in the mitochondria during apoptosis. The green-fluorescent dye JC (5,5,6,6 -tetrachloro- 1,1,3,3 -tetraethylbenzimidazolylcarbocyanine iodide, T-3168; Figure 23.13) 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 (Figure 12.21, Figure 12.22, Figure 23.14) that exhibit a broad excitation spectrum and an emission maximum at ~590 nm (Figure 23.15). 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 JC-1 has been used successfully to follow mitochondrial dysfunction in apoptotic hippocampal neurons 143 and opening of the mitochondrial permeability transition pore (MTP). 144,145 Our JC-9 dye (3,3 -dimethyl-α-naphthoxacarbocyanine iodide, D-22421, Figure 23.17) undergoes a similar potential-dependent spectral shift from a green-fluorescent product to a red-fluorescent aggregate (Figure 23.18) 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 (M-7512, Figure 12.4) 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. 151 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. 152 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 (M-7514, Section 12.2) result in improved discrimination of apoptotic and non-apoptotic cell populations. 146 Rhodamine 123 (R-302; FluoroPure grade, R-22420) is a cellpermeant, cationic, fluorescent dye that is readily sequestered by active mitochondria without inducing cytotoxic effects. 153 Uptake and equilibration of rhodamine 123 is rapid a few minutes compared to other membrane potential sensitive dyes, which may take 30 minutes or longer. 154 Although not aldehyde-fixable, rhodamine 123 allows for quick and easy detection of apoptotic cells. 155,156 Most carbocyanine dyes with short (C 1 C 6 ) alkyl chains stain mitochondria of live cells when used at low concentrations (~0.5 µm or ~0.1 µg/ml). DiOC 6 (3) (D-273) is a green-fluores- Figure Detection of apoptosis in SK-N-MC neuroblastoma cells. Following a six-hour exposure to hydrogen peroxide, cells were labeled with Hoechst (H-1399, H-3570, H-21492), tetramethylrhodamine ethyl ester (TMRE, T-669) and rhodamine 110, bis-l-aspartic acid amide (R-22122) for 15 minutes. Apoptotic cells show green cytosolic fluorescence resulting from cleavage of the rhodamine 110, bis-l-aspartic acid amide substrate by active caspase-3. The staining pattern of the Hoechst dye reveals that the majority of the rhodamine 110 positive cells also contain condensed or fragmented nuclei characteristic of apoptosis. Furthermore, the rhodamine 110 positive cells are also characterized by an absence of polarized mitochondria, as indicated by their failure to load the positively charged mitochondrial indicator TMRE. Image contributed by A.K. Stout and J.T. Greenamyre, Emory University. Section

10 cent 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. 157 The accumulation of both the methyl and ethyl esters of tetramethylrhodamine (TMRM, T-668; TMRE, T-669) in mitochondria and endoplasmic reticulum is driven by membrane potential. 158,159 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 160 and to investigate the mitochondrial permeability transition pore Calcein, a green-fluorescent dye that is formed inside cells that are loaded using calcein AM (C-1430, C-3099, C-3100; Section 15.2, Figure 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. 164 The transport of calcein through the MTP has been used to study the role of the MTP in apoptosis. 161,162,165 Nonyl acridine orange (A-1372) is reported to bind to cardiolipin in the inner mitochondrial membrane. Its fluorescence decreases as cardiolipin becomes oxidized or otherwise altered during apoptosis. 156, Our SYTO 16 green-fluorescent nucleic acid stain (S-7578) 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. 23,149 Apoptosis Assays Using Free Radical Probes The bcl-2 proto-oncogene product is reported to play a role in preventing apoptosis through its antioxidant properties. 17,169,170 Following an apoptotic signal, cells sustain progressive lipid peroxidation as detected with cis-parinaric acid (P-1901) that can be suppressed by bcl-2 overexpression. 170 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. 171,172 The reagent diphenyl-1- pyrenylphosphine (DPPP, D-7894) 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. 177 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. 178 The most common fluorogenic probe for detecting reactive oxygen species is 2,7 -dichlorodihydrofluorescein diacetate (H 2 DCFDA, D-399), which has been used to examine the effect of caspase-3 inhibitors on hydrogen peroxide production during apoptosis, 179 in apoptotic embryos 180 and in chemosensitive or chemoresistant cancer cells. 17 H 2 DCFDA can detect socalled necrotic zones containing cells under oxidative stress in tissues; 181 however, for this application we recommend our 5- (and-6)-chloromethyl-2,7 -dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA, C-6827; Figure 14.18), which has greater cell-membrane permeability and better cell retention of its green-fluorescent oxidation product. The acetoxymethyl ester of 2,7 -dichlorodihydrofluorescein diacetate (C-2938, Figure 15.79) is also more permeant to live cells and tissues and has been used to detect hydrogen peroxide in transplanted myoblasts. 182 As would be expected, the other major probes for reactive oxygen species dihydrorhodamine 123 (D-632, D-23806; Figure 15.19) and dihydroethidium (hydroethidine; D-1168, D-11347, D-23107), 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. 43,167 Dihydrocalcein AM (D-23805, Figure 15.4) is our newest scavenger of reactive oxygen species. The green-fluorescent dye calcein that is formed by intracellular oxidation (Figure 14.32) has cell retention that 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 183 (Section 19.3), although superoxide has also been implicated. 184,185 Probes such as 10-acetyl-3,7-dihydroxyphenoxazine (the Amplex Red reagent, A-12222, A-22177; Section 19.2) 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 Chapter 19. Apoptosis Assays Using Ion Indicators Figure C carboxy-2,7 -dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester). Significant changes in intracellular ph, Na +, K + and Ca 2+ concentrations accompany apoptosis. The role of acidification in apoptosis has been investigated using carboxy SNARF-1 AM acetate (C-1271, C-1272; Section 21.2) and BCECF AM 189 (B-1150, B-1170, B-3051; Section 21.2) cell-permeant ph indicators. 187 Low intracellular ph, as measured with the carboxy SNARF-1 ph indicator, and uptake of Hoechst have been shown to be correlated in partially apoptotic cell populations. 31 Plasma membrane depolarization and inactivation of the Na + /K + - ATPase early in apoptosis leads to an increase in intracellular Na + levels, as detected with SBFI AM (S-1263, Section 22.1), and an 656 Chapter 15 Assays for Cell Viability, Proliferation and Function

11 inhibition of K + uptake, as detected with 86 Rb + studies. 190 Changes in intracellular Ca 2+ levels may influence gene expression, as well as nuclease, protease and kinase activity Our extensive selection of Ca 2+ indicators, caged Ca 2+ reagents, Ca 2+ ionophores and Ca 2+ chelators (Chapter 20) may help to sort out the mechanism of Ca 2+ action in apoptosis. Apoptosis Assays Using Esterase Substrates Alterations in membrane permeability that occur during apoptosis have been monitored using nucleic acid stains (see above). These membrane changes may also affect the uptake and retention of our various general esterase substrates (Table 15.1) and substrates for other intracellular enzymes (Chapter 10). Results from staining apoptotic thymocytes with esterase substrates, however, showed significant variation depending on which probe was used. 25,58 Some of this variation undoubtedly resulted from differences in the ph sensitivity of the probes; thus, calcein AM (C-1430, C-3099, C-3100; Section 15.2), which has low ph sensitivity in the physiological ph range, may be the best reagent for detecting membrane permeability changes that accompany apoptosis. Calcein AM has been extensively used to detect the permeability transition of the mitochondrial membrane that apparently accompanies late stages of apoptosis. 152,164,199,200 Calcein AM has been recommended as a better marker for early apoptotic events than annexin V conjugates in NIH 3T3 fibroblasts. 201 Confocal laser-scanning microscopy of calcein AM labeled cells shows a large increase in nuclear fluorescence and cell shrinkage during the early stages of chromatin condensation and nuclear fragmentation. 201 Calcein AM staining also measures other important characteristics of apoptotic cells, including membrane blebbing and preservation of membrane integrity. An Apoptosis Assay that Measures the ATP:ADP Ratio Apoptotic cells are reported to have a relatively low ratio of ATP to ADP, apparently indicating decreased resynthesis of ATP in the mitochondria A very sensitive chemiluminescent assay that measures the average ATP:ADP ratio of cultured apoptotic cells based on the principles of our luciferin/luciferase-based ATP Determination Kit (A-22066; Section 10.3, Section 15.2) has been described. 205 References 1. J Immunol 132, 38 (1984); 2. Science 281, 1302 (1998); 3. Blood 85, 359 (1995); 4. Genes Dev 8, 2817 (1994); 5. Immunol Cell Biol 76, 1 (1998); 6. Cytometry 27, 1 (1997); 7. FASEB J 9, 1277 (1995); 8. Am J Pathol 146, 3 (1995); 9. Cell 74, 777 (1993); 10. J Pharm Sci 87, 1368 (1998); 11. Methods Cell Biol 41, 15 (1994); 12. Cytometry 35, 80 (1999); 13. Cell Mol Life Sci 53, 527 (1997); 14. J Immunol Methods 184, 39 (1995); 15. Blood 84, 1415 (1994); 16. Cytometry 27, 136 (1997); 17. Ann Surg Oncol 5, 287 (1998); 18. 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12 References continued (2000); 145. J Exp Med 191, 33 (2000); 146. Cytometry 35, 311 (1999); 147. Cytometry 25, 333 (1996); 148. Science 281, 2027 (1998); 149. Cytometry 27, 358 (1997); 150. J Histochem Cytochem 44, 1363 (1996); 151. J Cell Biol 141, 1243 (1998); 152. Exp Cell Res 246, 26 (1999); 153. Proc Natl Acad Sci U S A 77, 990 (1980); 154. Int Rev Cytol 122, 1 (1990); 155. Immunol Lett 61, 157 (1998); 156. Cytometry 24, 106 (1996); 157. J Biol Chem 272, (1997); 158. Biophys J 56, 1053 (1989); 159. Biophys J 53, 785 (1988); 160. J Biol Chem 274, 5654 (1999); 161. Am J Physiol 272, C1286 (1997); 162. Am J Physiol 273, C1783 (1997); 163. Biochem J 307, 99 (1995); 164. Biochim Biophys Acta 1366, 177 (1998); 165. Biophys J 76, 725 (1999); 166. Biochem Biophys Res Commun 269, 542 (2000); 167. J Cell Biol 138, 449 (1997); 168. Nature 389, 300 (1997); 169. Science 281, 1322 (1998); 170. Cell 75, 241 (1993); 171. Nature 378, 776 (1995); 172. J Immunol 155, 5133 (1995); 173. J Chromatogr 628, 31 (1993); 174. J Chromatogr 622, 153 (1993); 175. J Chromatogr 596, 197 (1992); 176. Anal Lett 21, 965 (1988); 177. FEBS Lett 474, 137 (2000); 178. J Immunol 156, 42 (1996); 179. J Cell Biol 273, (1998); 180. Hum Reprod 13, 998 (1998); 181. Exp Cell Res 238, 136 (1998); 182. J Immunol 159, 2522 (1997); 183. FEBS Lett 416, 175 (1997); 184. Neuroscience 86, 1109 (1998); 185. J Biol Chem 270, (1995); 186. J Immunol Methods 221, 43 (1998); 187. Proc Natl Acad Sci U S A 93, 654 (1996); 188. J Biol Chem 270, 3203 (1995); 189. J Biol Chem 270, 6235 (1995); 190. J Biol Chem 276, 4304 (2001); 191. Biochem Biophys Res Commun 214, 1130 (1995); 192. J Neurosci 15, 1172 (1995); 193. Biochim Biophys Acta 1223, 247 (1994); 194. Cell Calcium 16, 279 (1994); 195. Exp Cell Res 212, 84 (1994); 196. FASEB J 8, 237 (1994); 197. J Immunol 151, 5198 (1993); 198. Exp Cell Res 197, 43 (1991); 199. J Cell Biol 105, 2959 (1987); 200. Biophys J 74, 2129 (1998); 201. J Histochem Cytochem 46, 895 (1998); 202. Neuroscience 86, 279 (1998); 203. Exp Cell Res 226, 264 (1996); 204. Blood 84, 1613 (1994); 205. J Immunol Methods 240, 79 (2000). Data Table 15.5 Assays for Apoptosis Cat # MW Storage Soluble Abs EC Em Solvent Notes A L H 2 O, EtOH , H 2 O/DNA 1, 2 A F,L DMF, DMSO , H 2 O/DNA 1 A L DMSO, EtOH , MeOH A RR,L H 2 O , H 2 O/DNA A F,D DMSO , MeOH 3, 4 A F,D DMSO 339 7, MeOH 4, 5 A F,D DMSO , MeOH 3, 4 A F,D DMSO 340 8, MeOH 4, 5 C F,D,AA DMSO 291 5,700 none MeOH 6 C F,D,AA DMSO 287 9,100 none MeOH 6 C-7604 ~993 FF,L H 2 O , ph 8 7, 8 C-7614 ~908 FF,L H 2 O , ph 8 7, 8 C ~861 FF H 2 O <300 none 7, 8 D D,L DMSO , MeOH D F,D DMSO, EtOH ,000 none MeOH 6 D F,D,L,AA DMF, DMSO 289 7,100 none MeOH 9, 10 D FF,L,AA DMF, DMSO ,000 see Notes MeCN 9, 11 D F,D,LL MeCN ,000 none MeOH 12 D FF,L,AA DMF, DMSO ,000 see Notes MeCN 9, 11 D D,L DMSO, DMF , CHCl 3 13 D FF,D,L,AA DMSO ,000 see Notes MeCN 11, 14 D F,D DMSO 285 5,800 none MeCN 15 D F,D,L,AA DMSO 289 7,100 none MeOH 10, 14 E F,D,L DMSO , MeOH 3, 4, 16 E F,D,L DMSO ,000 none MeOH 16, 17 H L H 2 O, DMF , H 2 O/DNA 1, 18 H RR,L H 2 O , H 2 O/DNA 1, 8, 18 H L H 2 O, DMF , H 2 O/DNA 1, 18, 19 L L DMSO, EtOH , H 2 O/DNA 1 M F,D,L DMSO , MeOH P FF,LL,AA EtOH , MeOH 20 P F,D DMSO, MeOH <300 none P F,D,L DMSO , MeOH R F,D,L MeOH, DMF , MeOH R F,D DMSO, DMF ,000 none MeOH 17 R F,D DMSO, DMF ,000 none MeOH 17 R F,D DMSO, DMF ,000 none MeOH 17 R F,D DMSO, DMF ,000 none MeOH 17 R F,D,L MeOH, DMF , MeOH 19 S-7575 ~400 F,D,L DMSO , H 2 O/DNA S-7578 ~450 F,D,L DMSO , H 2 O/DNA 1, 8, 21, 22 S-7579 ~650 F,D,L DMSO , H 2 O/DNA T F,D,L DMSO, MeOH , MeOH T F,D,L DMSO, EtOH , MeOH T D,L DMSO, DMF , MeOH 23 Y F,D,L DMSO , H 2 O/DNA 1, 8, 21, 24 For definitions of the contents of this data table, see How to Use This Book on page viii. 658 Chapter 15 Assays for Cell Viability, Proliferation and Function

13 Notes 1. Spectra represent aqueous solutions of nucleic acid bound dye. EC values are derived by comparing the absorbance of the nucleic acid bound dye with that of free dye in a reference solvent (H 2 O or MeOH). 2. Acridine orange bound to RNA has Abs ~460 nm, Em ~650 nm (Methods Cell Biol 41, 401 (1994); Cytometry 2, 201 (1982)). 3. Peptidase cleavage of this substrate yields A-191 (Section 10.1). 4. Fluorescence of the unhydrolyzed substrate is very weak. 5. Enzymatic cleavage of this substrate yields 7-amino-4-trifluoromethylcoumarin: Abs = 376 nm (EC = 18,000 cm -1 M -1 ), Em = 480 nm in MeOH. 6. Dihydrofluorescein diacetates are colorless and nonfluorescent until both the acetates are hydrolyzed and the products are subsequently oxidized to fluorescein derivatives. The materials contain less than 0.1% of oxidized derivative when initially prepared. The end products from C-2938, C-6827 and D-399 are 2,7 -dichlorofluorescein derivatives with spectra similar to C-368 (Section 21.3). 7. The molecular weight (MW) of this product is approximate because the degree of hydration and/or salt form has not been conclusively established. 8. This product is supplied as a ready-made solution in the solvent indicated under Soluble. 9. This compound is susceptible to oxidation, especially in solution. Store solutions under argon or nitrogen. Oxidation appears to be catalyzed by illumination. 10. D-632 and D are essentially colorless and nonfluorescent until oxidized to R Dihydroethidium has blue fluorescence (Em ~420 nm) until oxidized to ethidium E-1305 (Section 15.2). The reduced dye does not bind to nucleic acids (FEBS Lett 26, 169 (1972)). 12. Oxidation product is strongly fluorescent. Em = 379 nm. Oxidation occurs rapidly in solution when illuminated. 13. JC-9 exhibits long-wavelength J-aggregate emission at ~635 nm in aqueous solutions and polarized mitochondria. 14. This product is supplied as a ready-made solution in DMSO with sodium borohydride added to inhibit oxidation. 15. D is colorless and nonfluorescent until the AM ester groups are hydrolyzed and the resulting leuco dye is subsequently oxidized. The final product is calcein C-481 (Section 14.3). 16. Data represent the substrate component of this kit. 17. Peptidase cleavage of this substrate yields R-6479 (Section 10.1). 18. MW is for the hydrated form of this product. 19. This product is specified to equal or exceed 98% analytical purity by HPLC. 20. Cis-parinaric acid is readily oxidized to nonfluorescent products. Use under N 2 or Ar except when oxidation is intended. Stock solutions should be prepared in deoxygenated solvents. Cis-parinaric acid is appreciably fluorescent in lipid environments and organic solvents but is nonfluorescent in water. 21. This product is essentially nonfluorescent except when bound to DNA or RNA. 22. MW: The preceding ~ symbol indicates an approximate value, not including counterions. 23. JC-1 forms J-aggregates with Abs/Em = 585/590 nm at concentrations above 0.1 µm in aqueous solutions (ph 8.0) (Biochemistry 30, 4480 (1991)). 24. Although this compound is soluble in water, preparation of stock solutions in water is not recommended because of possible adsorption onto glass or plastic. Product List 15.5 Assays for Apoptosis Cat # Product Name Unit Size A-1301 acridine orange... 1 g A-3568 acridine orange *10 mg/ml solution in water* ml A-1372 acridine orange 10-nonyl bromide (nonyl acridine orange) mg A aminoactinomycin D (7-AAD)... 1 mg A amino-4-chloromethylcoumarin, t-boc-l-leucyl-l-methionine amide (CMAC, t-boc-leu-met)... 5 mg A amino-4-methylcoumarin, N-CBZ-L-isoleucyl-L-glutamyl-L-threonyl-L-aspartic acid amide (Z-IETD-AMC)... 5 mg A amino-4-trifluoromethylcoumarin, N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide (Z-DEVD-AFC)... 5 mg A amino-4-trifluoromethylcoumarin, N-CBZ-L-isoleucyl-L-glutamyl-L-threonyl-L-aspartic acid amide (Z-IETD-AFC)... 5 mg A annexin V, Alexa Fluor 350 conjugate *100 assays* µl A annexin V, Alexa Fluor 488 conjugate *100 assays* µl A annexin V, Alexa Fluor 568 conjugate *100 assays* µl A annexin V, Alexa Fluor 594 conjugate *100 assays* µl A annexin V, Alexa Fluor 647 conjugate *100 assays* µl A annexin V, biotin-x conjugate *100 assays* µl A annexin V, fluorescein conjugate (FITC annexin V) *100 assays* µl A annexin V, Oregon Green 488 conjugate *100 assays* µl A APO-BrdU TUNEL Assay Kit *with Alexa Fluor 488 anti-brdu* *60 assays*... 1 kit C carboxy-2,7 -dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester)... 5 mg C (and-6)-chloromethyl-2,7 -dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA) *mixed isomers* *special packaging* x 50 µg C ChromaTide biotin-11-dutp *1 mm in TE buffer* µl C-7614 ChromaTide BODIPY FL-14-dUTP *1 mm in TE buffer* µl C-7604 ChromaTide fluorescein-12-dutp *1 mm in TE buffer* µl D-399 2,7 -dichlorodihydrofluorescein diacetate (2,7 -dichlorofluorescin diacetate; H 2 DCFDA) mg D-273 3,3 -dihexyloxacarbocyanine iodide (DiOC 6 (3)) mg D dihydrocalcein, AM *special packaging* x 50 µg D-1168 dihydroethidium (hydroethidine) mg D dihydroethidium (hydroethidine) *special packaging* x 1 mg D dihydroethidium (hydroethidine) *5 mm stabilized solution in DMSO*... 1 ml D-632 dihydrorhodamine mg D dihydrorhodamine 123 *5 mm stabilized solution in DMSO*... 1 ml D ,3 -dimethyl-α-naphthoxacarbocyanine iodide (JC-9; DiNOC 1 (3))... 5 mg D-7894 diphenyl-1-pyrenylphosphine (DPPP)... 5 mg E EnzChek Caspase-3 Assay Kit #1 *Z-DEVD-AMC substrate* *500 assays*... 1 kit E EnzChek Caspase-3 Assay Kit #2 *Z-DEVD-R110 substrate* *500 assays*... 1 kit Section

14 Product List 15.5 Assays for Apoptosis continued Cat # Product Name Unit Size H-3570 Hoechst *10 mg/ml solution in water* ml H-1399 Hoechst 33342, trihydrochloride, trihydrate mg H Hoechst 33342, trihydrochloride, trihydrate *FluoroPure grade* mg L-7595 LDS mg M-7512 MitoTracker Red CMXRos *special packaging* x 50 µg P-1901 cis-parinaric acid *special packaging* x 10 mg P-6542 pepstatin A (iso-valeryl-l-val-l-val-sta-l-ala-sta) mg P pepstatin A, BODIPY FL conjugate µg R rhodamine 110, bis-(l-aspartic acid amide), trifluoroacetic acid salt... 1 mg R rhodamine 110, bis-(n-cbz-l-aspartyl-l-glutamyl-l-valyl-l-aspartic acid amide) (Z-DEVD-R110) *bulk packaging* mg R rhodamine 110, bis-(n-cbz-l-isoleucyl-l-glutamyl-l-threonyl-l-aspartic acid amide) (Z-IETD-R110)... 2 mg R rhodamine 110, bis-(n-cbz-l-isoleucyl-l-glutamyl-l-threonyl-l-aspartic acid amide) (Z-IETD-R110) *bulk packaging* mg R-302 rhodamine mg R rhodamine 123 *FluoroPure grade* mg S-7575 SYTO 13 green fluorescent nucleic acid stain *5 mm solution in DMSO* µl S-7578 SYTO 16 green fluorescent nucleic acid stain *1 mm solution in DMSO* µl S-7579 SYTO 17 red fluorescent nucleic acid stain *5 mm solution in DMSO* µl T ,5,6,6 -tetrachloro-1,1,3,3 -tetraethylbenzimidazolylcarbocyanine iodide (JC-1; CBIC 2 (3))... 5 mg T-669 tetramethylrhodamine, ethyl ester, perchlorate (TMRE) mg T-668 tetramethylrhodamine, methyl ester, perchlorate (TMRM) mg V Vybrant Apoptosis Assay Kit #1 *Alexa Fluor 488 annexin V/SYTOX Green* *50 assays*... 1 kit V Vybrant Apoptosis Assay Kit #2 *Alexa Fluor 488 annexin V/propidium iodide* *50 assays*... 1 kit V Vybrant Apoptosis Assay Kit #3 *FITC annexin V/propidium iodide* *50 assays*... 1 kit V Vybrant Apoptosis Assay Kit #4 *YO-PRO -1/propidium iodide* *200 assays*... 1 kit V Vybrant Apoptosis Assay Kit #5 *Hoechst 33342/propidium iodide* *200 assays*... 1 kit V Vybrant Apoptosis Assay Kit #6 *biotin-x annexin V/Alexa Fluor 350 streptavidin/propidium iodide* *50 assays*... 1 kit V Vybrant Apoptosis Assay Kit #7 *Hoechst 33342/YO-PRO -1/propidium iodide* *200 assays*... 1 kit Y-3603 YO-PRO -1 iodide (491/509) *1 mm solution in DMSO*... 1 ml 15.6 Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione Cell Adhesion Assays The fundamental role of cell cell and cell matrix adhesion in the morphology and development of organisms, organs and tissues has made identification of molecular mediators of cell adhesion an important research focus in cell biology and immunology. 1 3 The useful review by Löster and Hortstkorte describes a number of different assays that can detect cell adhesion. 4 In a typical fluorescence-based cell adhesion assay, unlabeled cell monolayers in multiwell plates are incubated with fluorescently labeled cells and then washed to separate the adherent and nonadherent populations. Cell adhesion can then be determined simply by correlating the retained fluorescence with cell number. An ideal fluorescent marker will retain proportionality between fluorescence and cell number and introduce minimal interference with the cell adhesion process. Because adhesion is a cell-surface phenomenon, cytoplasmic markers that can be passively loaded are preferable to compounds that label cell-surface molecules, provided that they are retained in the cell for the duration of the experiment or their leakage rate can be independently measured. Adhesion of fluorescent dye labeled cells to matrices such as bone 5 can be directly observed by fluorescence microscopy using cells loaded with the permeant live-cell tracers described in Section 14.2 and Section 15.2 or the lipophilic dyes in Section Alternatively, high molecular weight, cell-impermeant, fluorescent dextrans (Section 14.5) have been used to define the area outside of adherent cells, with the adherent cells themselves remaining unstained. 6 This same negative-staining method can also be used to assess cell spreading and progress toward confluency. Cell Adhesion Assays Using Enzyme Substrates Essentially all of the esterase substrates in Table 15.1 useful for monitoring cell viability can also be used for studying cell adhesion. As with cell viability studies, calcein AM (C-1430, C-3099, C-3100; Figure 15.2) appears to best satisfy the criteria for assaying cell adhesion 7 9 and to have the least effect on cell viability and other cell functions. 7 The results obtained with leukocyte adhesion assays using calcein AM correlate well with those obtained with 51 Cr assays, 10,11 but the calcein AM protocols are more rapid and avoid the special handling required for use of 660 Chapter 15 Assays for Cell Viability, Proliferation and Function