15.5 Assays for Apoptosis
Apoptosis (programmed cell death) is the genetically con-trolled 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 charac-terized 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 Anti-cancer drug candidates failing to induce apoptosis are likely to have decreased clinical efficacy,17 making apoptosis assays im-portant 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 poten-tially be used as tracers for phagocytosis,18 a cell process that is discussed in Section 16.1.
Apoptosis Assays Using Nucleic Acid Stains
The characteristic breakdown of the nucleus during apoptosis comprises collapse and fragmentation of the chromatin, degrada-tion 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 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.21 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 (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 apop-totic 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-fluores-cent nucleic acid stain (S-7579) than with either fluorescein-labeled annexin V or propidium iodide.24
• Hoechst 33342 (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 25–27 (E-1305, E-3565; Section 8.1) are excluded. Later stages of apoptosis are accompanied by an increase in membrane perme-ability, 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 fluores-cence 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 33342 uptake in partially apoptotic cell populations is correlated with low intracellular pH, as mea-sured 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 popu-lation could be selectively detected using propidium iodide to make this a three-color assay.
Figure 15.65 Absorption and fluorescence emission spectra of YO-PRO-1 (Y-3603) bound to DNA.
• 7-Aminoactinomycin D (7-AAD, A-1310) 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.34–38 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 micro-scopy.39 7-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 compro-mised membrane of apoptotic cells; photolysis results in covalent labeling of intra-cellular nucleic acids that persists through fixation and permeabilization.49
Figure 15.66 DNA extracts from camptothecin-treated 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 prepara-tions were gifts of Zbigniew Darzynkiewicz, Can-cer 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) *
Probe for Necrotic Cells
(Abs/Em) *
Number of Assays Kit Features
V-13240 Vybrant Apoptosis Assay Kit #1 Alexa Fluor 488 annexin V (495/519) SYTOX Green nucleic acid stain (504/523)
50 flow cytometry assays, each containing 2 ¥ 105 to 1 ¥ 106 cells
in a 1 mL volume
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.
V-13241 Vybrant Apoptosis Assay Kit #2 Alexa Fluor 488 annexin V (495/519) Propidium iodide (535/617) 50 flow cytometry assays, each containing 2 ¥ 105 to 1 ¥ 106 cells
in a 1 mL volume
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. V-13242 Vybrant Apoptosis Assay Kit #3 FITC annexin V (494/519) Propidium iodide (535/617) 50 flow cytometry assays, each containing 2 ¥ 105 to 1 ¥ 106 cells
in a 1 mL volume
Kit #3 is identical to Kit #2 except that it contains the fluorescein conjugate of annexin V.
V-13243 Vybrant Apoptosis Assay Kit #4 YO-PRO-1 dye (491/509) Propidium iodide (535/617) 200 flow cytometry assays, each containing 2 ¥ 105 to 1 ¥ 106 cells
in a 1 mL volume
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. V-13244 Vybrant Apoptosis Assay Kit #5 Hoechst 33342 (346/480) Propidium iodide (535/617) 200 flow cytometry assays, each containing 2 ¥ 105 to 1 ¥ 106 cells
in a 1 mL volume
Kit #5 uses Hoechst 33342 in combination with propidium iodide to distinguish between the condensed chromatin of apoptotic cells and the looser chromatin structure in live cells.
V-23200 Vybrant Apoptosis Assay Kit #6
Biotin-X annexin V and Alexa Fluor 350 streptavidin (345/442)
Propidium iodide (535/617)
200 flow cytometry assays, each containing 2 ¥ 105 to 1 ¥ 106 cells
in a 1 mL volume
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. V-23201 Vybrant Apoptosis Assay Kit #7 Hoechst 33342 (346/480) and YO-PRO-1 dye (491/509) Propidium iodide (535/617) 200 flow cytometry assays, each containing 2 ¥ 105 to 1 ¥ 106 cells
in a 1 mL volume
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.
Figure 15.68 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 apop-totic and necrotic cells were detected with propidi-um iodide (red) (P-1304, P-3566, P-21493). Figure 15.67 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) fol-lowed 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 apopto-sis seen in the control cells (top panel). V = viable cells, N = necrotic cells.
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). Electro-phoresis of apoptotic cells in an agarose gel matrix results in the formation of dis-tinctive “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 mem-branes 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 effec-tively 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 summa-rized in Table 15.4.
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 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 (Table 15.4). The staining pattern resulting from the simulta-neous 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 33342 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 15.4.
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 provid-ed 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 exci-tation of all three dyes or with a combination of UV exciexci-tation for the Hoechst 33342 dye and excitation by the 488 nm spectral line of the argon-ion laser. Differences in the inten-sity 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 15.4.
Figure 15.70 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 In-stitute, New York Medical College.
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 iden-tify specific sequences with damaged DNA.63
Comet assays have traditionally been performed using ethidi-um 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 trans-ferase dUTP nick end labeling) assays are widely used for detect-ing 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 la-beled nucleotides to the 3′-hydroxyl ends of these DNA
frag-Figure 15.69 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 H2O2
and immobilized onto a Trevigen CometSlide for analysis. The cells were gently lysed, washed and treated with endonuclease. Slides were subjected to electro-phoresis in alkaline electroelectro-phoresis buffer and stained with SYBR Green I stain.
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 70–73 (Section 7.6, Table 7.17). However, a more direct approach for detecting DNA strand breaks in apoptot-ic 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 chro-matin 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 character-istic 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 pa-tients.77–80 DNA fragmentation is also associated with
amyo-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.84–86 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 apopto-sis,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 nu-cleases, 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 cy-tometry applications, though it may also be adapted for use with fluorescence micros-copy. 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
Sufficient reagents are provided for approximately 60 assays of 1 mL samples, each containing 1–2 × 106 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 anti-coagulant annexin V is a 35–36 kilodalton, Ca2+-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 intermedi-ate stages of apoptosis. Nuclear fragmentation, mitochondrial membrane potential flux and caspase-3 activation apparently precede phosphatidylserine “flipping” during apopto-sis, 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, confo-cal or epifluorescence microscopy and, like antibody staining, can be used in combina-tion with other dyes, including nucleic acid stains, to accurately assess mixed populacombina-tions
Figure 15.71 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 ap-optosis are detected by TUNEL and fluoresce green, while necrotic cells are stained with red-fluorescent propidium iodide.
Figure 15.72 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 Vy-brant Apoptosis Assay Kit #2 (V-13241), followed by flow cytometric analysis. Note that the camp-tothecin-treated cells (bottom panel) have a signifi-cantly higher percentage of apoptotic cells (indicat-ed by an “A”) than the basal level of apoptosis seen in the control cells (top panel). V = viable cells, N = necrotic cells.
of apoptotic and nonapoptotic cells.94 Our annexin V conjugates are available as stand-alone reagents, each suitable for at least 100 flow cytometric assays or many more imag-ing assays, or in several variations of our Vybrant Apoptosis Assay Kits (Table 15.4). Our annexin V conjugates include:
• 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 photo-stable 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 popula-tions.14,15,24,92,94 Fluorescein annexin V is frequently used in combination with pro-pidium 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 spec-trally similar to the fluorescein annexin V conjugate but is brighter and more photo-stable (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 excita-tion sources for detecexcita-tion of apoptotic cells by either flow cytometry or microscopy. • Alexa Fluor 350 annexin V (A-23202) can be excited in the ultraviolet and has
bright-blue 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). Figure 15.74 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 Vy-brant Apoptosis Assay Kit #1 (V-13240), followed by flow cytometric analysis. Note that the camp-tothecin-treated cells (green line) have a signifi-cantly higher percentage of apoptotic cells (inter-mediate green fluorescence) than the basal level of apoptosis seen in the control cells (blue line). Figure 15.73 Jurkat human T-cell leukemia cells treated with 1 µM camptothecin. The externalized phosphatidylserine, a characteristic of early-stage ap-optotic 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.
Table 15.5 Fluorogenic substrates for caspase activity.
Substrate *† Enzymes Cat #
Z-DEVD-AMC Caspase-3, Caspase-7 E-13183 Z-DEVD-R110 Caspase-3, Caspase-7 E-13184 R-22120 Z-DEVD-AFC Caspase-3, Caspase-7 A-22121 R110, bis-L-aspartic acid amide (D2-R110) Caspase-3, Caspase-7 R-22122
Z-IETD-AFC Caspase-8 A-22128
Z-IETD-AMC Caspase-8 A-22127
Z-IETD-R110 Caspase-8 R-22125
R-22126
* 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 R110.
Our fluorogenic caspase substrates are
available in bulk from Molecular Probes
for high-throughput screening
applica-tions. Contact [email protected] for
more information.
Vybrant Apoptosis Assay Kit #1
With the Vybrant Apoptosis Assay Kit #1 (V-13240), apoptot-ic cells are detected on the basis of the externalization of phos-phatidylserine. This kit contains recombinant annexin V conju-gated 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 argon-ion laser for excitatargon-ion. 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 sum-marized in Table 15.4.
Vybrant Apoptosis Assay Kit #2
Like the Vybrant Apoptosis Kit #1, our Vybrant Apoptosis Assay Kit #2 (V-13241) detects the externalization of phosphati-dylserine 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 popu-lation with Alexa Fluor 488 annexin V and propidium iodide in the provided binding buffer, apoptotic cells show green fluores-cence, dead cells show red and green fluoresfluores-cence, 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 compo-nents, number of assays and assay principles are summarized in Table 15.4.
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, num-ber of assays and assay principles are summarized in Table 15.4.
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 fluores-cently label the bound annexin V. Finally, propidium iodide is added to detect necrotic cells. Apoptotic cells show blue fluores-cence, 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 propidi-um 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 15.4.
Apoptosis Assays Based on Protease Activity
Caspases
Caspases comprise a key component of the apoptotic machin-ery 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 anoth-er protease.101
Caspase-3 Substrates and Assay Kits
Caspase-3 is a key effector in the apoptosis pathway, amplify-ing 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-R110 106–108 (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 quanti-tate 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
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-R110 107–109 (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 absorp-tion 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 mono-amide 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 fluores-cein, 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 puri-fied 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-L -aspartic acid amide of R110 (D2-R110, R-22122), which contains R110 flanked by aspartic acid residues (Table 15.5). D2-R110 does not appear to require any invasive techniques such as osmotic shock to gain entrance into the cytoplasm (Figure 15.78). It may
Figure 15.75 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 tempera-ture, 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 15.76 E-13183 Z-DEVD-AMC substrate included in the EnzChek Caspase-3 Assay Kit #1.
Figure 15.77 E-13184 Z-DEVD-R110 substrate included in the EnzChek Caspase-3 Assay Kit #2.
serve as a substrate for a variety of apoptosis-related proteases, including caspase-3 and caspase-7.107
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 caspas-es, such as caspase-3.115 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-R110 109 (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 report-ed to inhibit apoptosis in microglia, lymphoid cells and HeLa cells.119–121 Consequently, our cell-permeant BODIPY FL pep-statin derivative (P-12271), 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.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 prolifera-tion.121,124 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 caspas-es.125–127 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 trans-forming the substrate into a membrane-impermeant probe. Subse-quent 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.131–133 This mito-chondria disruption includes changes in the membrane potential and alterations to the oxidation–reduction potential of the mito-chondria. 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 respira-tory chain and then the release of cytochrome c into the cyto-sol.134,135 Molecular Probes has available several unique reagents for studying changes in the mitochondria during apoptosis. • The green-fluorescent dye JC-1 136–139(5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide, T-3168; Figure 23.13) exists as a monomer at low concentra-tions or at low membrane potential. However, at higher con-centrations — aqueous solutions above 0.1 µM — or at higher membrane potentials, JC-1 forms red-fluorescent “J-aggre-gates” (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 mitochon-drial membrane potential that occur as a result of apopto-sis.140–142 JC-1 has been used successfully to follow mitochon-drial 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-fluores-cent aggregate (Figure 23.18) and is likely to be similarly useful for detecting apoptotic cells by both imaging and flow cytome-try. 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.146–150 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 revers-ible 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 discrimina-tion of apoptotic and non-apoptotic cell populadiscrimina-tions.146 • Rhodamine 123 (R-302; FluoroPure grade, R-22420) is a
cell-permeant, 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 (C1–C6) alkyl chains stain mitochondria of live cells when used at low concentrations (~0.5 µM or ~0.1 µg/mL). DiOC6(3) (D-273) is a
green-fluores-Figure 15.78 Detection of apoptosis in SK-N-MC neuroblastoma cells. Follow-ing a six-hour exposure to hydrogen peroxide, cells were labeled with Hoechst 33342 (H-1399, H-3570, H-21492), tetramethylrhodamine ethyl ester (TMRE, T-669) and rhodamine 110, bis-L-aspartic acid amide (R-22122) for 15
min-utes. 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 33342 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.
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 tetra-methylrhodamine (TMRM, T-668; TMRE, T-669) in mito-chondria 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 mitochon-drial permeability transition pore.161–163
• 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 permeabili-ty 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 apop-tosis.161,162,165
• Nonyl acridine orange (A-1372) is reported to bind to cardio-lipin in the inner mitochondrial membrane. Its fluorescence decreases as cardiolipin becomes oxidized or otherwise altered during apoptosis.156,166–168
• 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 intracellularFigure 15.79 C-2938 6-carboxy-2′,7′-dichlorodihydrofluorescein diace-tate, di(acetoxymethyl ester).
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.173–176 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 perox-ide, and by sodium azperox-ide, 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 diace-tate (H2DCFDA, 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 chemosensi-tive or chemoresistant cancer cells.17 H
2DCFDA can detect so-called “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-H2DCFDA, 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 apop-totic 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 superox-ide 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 apop-tosis. All of our probes for detecting reactive oxygen species are described in Chapter 19.
Apoptosis Assays Using Ion Indicators
Significant changes in intracellular pH, Na+, K+ and Ca2+ concentrations accompany apoptosis. The role of acidification in apoptosis has been investigated using carboxy SNARF-1 AM acetate 186–188 (C-1271, C-1272; Section 21.2) and BCECF AM 189 (B-1150, B-1170, B-3051; Section 21.2) cell-permeant pH indi-cators.187 Low intracellular pH, as measured with the carboxy SNARF-1 pH indicator, and uptake of Hoechst 33342 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
inhibition of K+ uptake, as detected with 86Rb+ studies.190 Chang-es in intracellular Ca2+ levels may influence gene expression, as well as nuclease, protease and kinase activity.191–198 Our extensive selection of Ca2+ indicators, caged Ca2+ reagents, Ca2+ ionophores and Ca2+ chelators (Chapter 20) may help to sort out the mecha-nism of Ca2+ 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 beenexten-sively used to detect the permeability transition of the mito-chondrial 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 conju-gates in NIH 3T3 fibroblasts.201 Confocal laser-scanning mi-croscopy 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 Cal-cein AM staining also measures other important characteristics of apoptotic cells, including membrane blebbing and preserva-tion 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.202–204 A very sensitive chemiluminescent assay that measures the average ATP:ADP ratio of cultured apop-totic 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
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Chem 270, 6235 (1995); 190. J Biol Chem 276, 4304 (2001); 191. Biochem Biophys Res Com-mun 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-1301 301.82 L H2O, EtOH 500 53,000 526 H2O/DNA 1, 2
A-1310 1270.45 F,L DMF, DMSO 546 25,000 647 H2O/DNA 1
A-1372 472.51 L DMSO, EtOH 495 84,000 519 MeOH
A-3568 301.82 RR,L H2O 500 53,000 526 H2O/DNA
A-6520 554.10 F,D DMSO 330 13,000 403 MeOH 3, 4
A-22121 821.72 F,D DMSO 339 7,900 435 MeOH 4, 5
A-22127 767.79 F,D DMSO 326 17,000 392 MeOH 3, 4
A-22128 821.76 F,D DMSO 340 8,200 434 MeOH 4, 5
C-2938 675.43 F,D,AA DMSO 291 5,700 none MeOH 6
C-6827 577.80 F,D,AA DMSO 287 9,100 none MeOH 6
C-7604 ~993 FF,L H2O 496 68,000 523 pH 8 7, 8
C-7614 ~908 FF,L H2O 504 68,000 513 pH 8 7, 8
C-11411 ~861 FF H2O <300 none 7, 8
D-273 572.53 D,L DMSO 484 154,000 501 MeOH
D-399 487.29 F,D DMSO, EtOH 258 11,000 none MeOH 6
D-632 346.38 F,D,L,AA DMF, DMSO 289 7,100 none MeOH 9, 10
D-1168 315.42 FF,L,AA DMF, DMSO 355 14,000 see Notes MeCN 9, 11
D-7894 386.43 F,D,LL MeCN 358 29,000 none MeOH 12
D-11347 315.42 FF,L,AA DMF, DMSO 355 14,000 see Notes MeCN 9, 11
D-22421 532.38 D,L DMSO, DMF 522 143,000 535 CHCl3 13
D-23107 315.42 FF,D,L,AA DMSO 355 14,000 see Notes MeCN 11, 14
D-23805 1068.95 F,D DMSO 285 5,800 none MeCN 15
D-23806 346.38 F,D,L,AA DMSO 289 7,100 none MeOH 10, 14
E-13183 767.74 F,D,L DMSO 326 16,000 387 MeOH 3, 4, 16
E-13184 1515.46 F,D,L DMSO 232 52,000 none MeOH 16, 17
H-1399 615.99 L H2O, DMF 350 45,000 461 H2O/DNA 1, 18
H-3570 615.99 RR,L H2O 350 45,000 461 H2O/DNA 1, 8, 18
H-21492 615.99 L H2O, DMF 350 45,000 461 H2O/DNA 1, 18, 19
L-7595 471.98 L DMSO, EtOH 543 46,000 712 H2O/DNA 1
M-7512 531.52 F,D,L DMSO 578 116,000 599 MeOH
P-1901 276.42 FF,LL,AA EtOH 303 76,000 416 MeOH 20
P-6542 685.90 F,D DMSO, MeOH <300 none
P-12271 1044.14 F,D,L DMSO 504 86,000 511 MeOH
R-302 380.83 F,D,L MeOH, DMF 507 101,000 529 MeOH
R-22120 1515.46 F,D DMSO, DMF 232 52,000 none MeOH 17
R-22122 788.57 F,D DMSO, DMF 232 55,000 none MeOH 17
R-22125 1515.55 F,D DMSO, DMF 232 52,000 none MeOH 17
R-22126 1515.55 F,D DMSO, DMF 232 52,000 none MeOH 17
R-22420 380.83 F,D,L MeOH, DMF 507 101,000 529 MeOH 19
S-7575 ~400 F,D,L DMSO 488 74,000 509 H2O/DNA
S-7578 ~450 F,D,L DMSO 488 42,000 518 H2O/DNA 1, 8, 21, 22
S-7579 ~650 F,D,L DMSO 621 88,000 634 H2O/DNA
T-668 500.93 F,D,L DMSO, MeOH 549 115,000 573 MeOH
T-669 514.96 F,D,L DMSO, EtOH 549 109,000 574 MeOH
T-3168 652.23 D,L DMSO, DMF 514 195,000 529 MeOH 23
Y-3603 629.32 F,D,L DMSO 491 52,000 509 H2O/DNA 1, 8, 21, 24
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 (H2O 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-23806 are essentially colorless and nonfluorescent until oxidized to R-302.
11. 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-23805 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 N2 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* ... 10 mL A-1372 acridine orange 10-nonyl bromide (nonyl acridine orange) ... 100 mg A-1310 7-aminoactinomycin D (7-AAD) ... 1 mg A-6520 7-amino-4-chloromethylcoumarin, t-BOC-L-leucyl-L-methionine amide (CMAC, t-BOC-Leu-Met) ... 5 mg
A-22127 7-amino-4-methylcoumarin, N-CBZ-L-isoleucyl-L-glutamyl-L-threonyl-L-aspartic acid amide (Z-IETD-AMC) ... 5 mg
A-22121 7-amino-4-trifluoromethylcoumarin, N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide (Z-DEVD-AFC) ... 5 mg
A-22128 7-amino-4-trifluoromethylcoumarin, N-CBZ-L-isoleucyl-L-glutamyl-L-threonyl-L-aspartic acid amide (Z-IETD-AFC) ... 5 mg
A-23202 annexin V, Alexa Fluor® 350 conjugate *100 assays* ...
500 µL A-13201 annexin V, Alexa Fluor® 488 conjugate *100 assays* ...
500 µL A-13202 annexin V, Alexa Fluor® 568 conjugate *100 assays* ...
500 µL A-13203 annexin V, Alexa Fluor® 594 conjugate *100 assays* ...
500 µL A-23204 annexin V, Alexa Fluor® 647 conjugate *100 assays* ...
500 µL A-13204 annexin V, biotin-X conjugate *100 assays* ... 500 µL A-13199 annexin V, fluorescein conjugate (FITC annexin V) *100 assays* ... 500 µL A-13200 annexin V, Oregon Green® 488 conjugate *100 assays* ...
500 µL A-23210 APO-BrdU™ TUNEL Assay Kit *with Alexa Fluor® 488 anti-BrdU* *60 assays* ... 1 kit
C-2938 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) ... 5 mg C-6827 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) *mixed isomers*
*special packaging* ... 20 x 50 µg C-11411 ChromaTide™ biotin-11-dUTP *1 mM in TE buffer* ... 25 µL C-7614 ChromaTide™ BODIPY® FL-14-dUTP *1 mM in TE buffer* ...
25 µL C-7604 ChromaTide™ fluorescein-12-dUTP *1 mM in TE buffer* ... 25 µL D-399 2′,7′-dichlorodihydrofluorescein diacetate (2′,7′-dichlorofluorescin diacetate; H2DCFDA) ... 100 mg
D-273 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)) ... 100 mg
D-23805 dihydrocalcein, AM *special packaging* ... 20 x 50 µg D-1168 dihydroethidium (hydroethidine) ... 25 mg D-11347 dihydroethidium (hydroethidine) *special packaging* ... 10 x 1 mg D-23107 dihydroethidium (hydroethidine) *5 mM stabilized solution in DMSO* ... 1 mL D-632 dihydrorhodamine 123 ... 10 mg D-23806 dihydrorhodamine 123 *5 mM stabilized solution in DMSO* ... 1 mL D-22421 3,3′-dimethyl-α-naphthoxacarbocyanine iodide (JC-9; DiNOC1(3)) ... 5 mg
D-7894 diphenyl-1-pyrenylphosphine (DPPP) ... 5 mg E-13183 EnzChek® Caspase-3 Assay Kit #1 *Z-DEVD-AMC substrate* *500 assays* ... 1 kit