Section 15.5 - Assays for Apoptosis. Molecular Probes Chapter

Section 15.5 - Assays for Apoptosis. Molecular Probes Chapter

Apoptosis (programmed cell death) is the genetically controlled ablation of cells during normal development. Inappropriately regulated apoptosis is implicated in disease states such as Alzheimer's disease, stroke and cancer. Apoptosis is distinct from necrosis in both the biochemical and the morphological changes that occur. 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 in all systems; 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. Anti-cancer drug candidates failing to induce apoptosis are likely to have decreased clinical efficacy, 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, a cell process that is discussed in Section 16.1.

Apoptosis Assays Using Nucleic Acid Stains

DNA Stains For Detection of Apoptotic Cells

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

* Our YO-PRO-1 (Y3603) nucleic acid stain is the basis of an important assay for apoptotic cells that is compatible with both fluorescence microscopy and flow cytometry. Selective uptake of YO-PRO-1 by apoptotic cells of a dexamethasone-treated population of thymocytes, an irradiated peripheral blood mononuclear cell population and a growth factor–depleted tumor B cell line was confirmed by cell sorting. Unlike Hoechst 33342 staining, YO-PRO-1 staining had no effect on the ability of stained T cells to proliferate. Moreover, the visible-light absorption of the YO-PRO-1 stain () eliminates the need for UV excitation capabilities in flow cytometry. YO-PRO-1 is the key reagent in our Vybrant Apoptosis Assay Kits #4 and #7 (V13243, V23201, see below), which provide the reagents and tested protocols for combination flow cytometric apoptosis and necrosis assays.

* Some of our cell-permeant, green-fluorescent SYTO dyes, including the SYTO 13 and SYTO 16 nucleic acid stains (S7575, S7578), are proving useful for distinguishing apoptotic neuronal cells and apoptotic thymocytes. Our SYTO Fluorescent Nucleic Acid Stain Sampler Kits (S7554, S7572, S11340, S11350, S11360; 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 (S7579) than with either fluorescein-labeled annexin V or propidium iodide.

* Hoechst 33342 (H1399, H3570; FluoroPure Grade, H21492) is readily taken up by cells during the initial stages of apoptosis, whereas cell-impermeant dyes such as propidium iodide (P1304MP, P3566, P21493; Section 8.1) and ethidium bromide (E1305, E3565; Section 8.1) are excluded. Later stages of apoptosis are accompanied by an increase in membrane permeability, which allows propidium iodide to enter cells. Thus, a combination of Hoechst 33342 and propidium iodide has been extensively used for simultaneous flow cytometric and fluorescence imaging analysis of the stages of apoptosis and cell-cycle distribution. Our Vybrant Apoptosis Assay Kit #5 (V13244, see below) is based on these reagents and our Vybrant Apoptosis Assay Kit #7 (V23201, see below) adds the YO-PRO-1 nucleic acid to selectively determine the apoptotic cell population in a three-color experiment.

* The rate of Hoechst 33342 uptake in partially apoptotic cell populations is correlated with low intracellular pH, as measured with our carboxy SNARF-1 pH indicator (C1271, C1272; Section 21.2).

* Hoechst 33342, which selectively stains nuclei of apoptotic cells blue fluorescent, has also been used in combination with calcein AM (C1430, C3099, C3100MP; Section 15.2), which stains all cells that have intact membranes — even apoptotic cells — green fluorescent. Presumably the dead-cell population could be selectively detected using propidium iodide to make this a three-color assay.

* 7-Aminoactinomycin D (7-AAD, A1310) has been used alone or in combination with Hoechst 33342 to separate populations of live cells, early apoptotic cells and late apoptotic cells by flow cytometry. The staining pattern of 7-AAD is retained following cell fixation, and its unusually large Stokes shift is advantageous when simultaneously staining with cell-surface labels. 7-AAD staining has also been used to detect apoptotic cells by their characteristic morphology using fluorescence microscopy. 7-AAD has also been used in combination with the green-fluorescent SYTO 16 nucleic acid stain (S7578) to detect early stages of apoptosis that could not be detected by 7-AAD alone.

* The cell-permeant nucleic acid stain LDS 751 (L7595) has been used to discriminate intact nucleated cells from nonnucleated cells and cells with damaged nuclei, as well as to differentiate apoptotic cells from nonapoptotic cells.

* Acridine orange (A1301, A3568) exhibits metachromatic fluorescence that is sensitive to DNA conformation, making it a useful probe for detecting apoptotic cells. When analyzed by flow cytometry, apoptotic cells stained by acridine orange show reduced green fluorescence and enhanced red fluorescence in comparison to normal cells.

* DAPI (D1306, D21490; Section 8.1) and sulforhodamine 101 (S359, Section 14.3) can be used together in fixed apoptotic cells to reveal concomitant breakdown of proteins and DNA.

* The excited-state lifetime of ethidium homodimer-2 (E3599, Section 8.1) has been shown to be different in populations of aldehyde-fixed apoptotic and nonapoptotic cells.

* Ethidium monoazide (E1374, Section 15.2) passes through the partially compromised membrane of apoptotic cells; photolysis results in covalent labeling of intracellular nucleic acids that persists through fixation and permeabilization.

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

Vybrant Apoptosis Assay Kit #4

Our Vybrant Apoptosis Assay Kit #4 (V13243) detects apoptosis 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. 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 15.80) and fluorescence microscopy (). We have optimized Our Vybrant Apoptosis Assay Kits using Jurkat cells, a human T-cell leukemia clone, treated with camptothecin to induce apoptosis. Some modifications may be required for use with other cell types. The kit components, number of assays and assay principles are summarized in Table 15.4.

Vybrant Apoptosis Assay Kits #5 and #7

The Vybrant Apoptosis Assay Kit #5 (V13244) provides a rapid and convenient assay for apoptosis based upon fluorescence detection of the compacted state of the chromatin in apoptotic cells. This kit contains ready-to-use solutions of the blue-fluorescent Hoechst 33342 dye (excitation/emission maxima ~350/461 nm when bound to DNA), which stains the condensed chromatin of apoptotic cells more brightly than the chromatin of nonapoptotic cells, and the red-fluorescent propidium iodide (excitation/emission maxima ~535/617 nm when bound to DNA), which is permeant only to dead cells with compromised membranes (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. 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 provided as separate solutions to facilitate optimization of the assay for the cell line under study and the equipment available. However, once optimized, the assay can be completed using simultaneous staining with a mixture of the three nucleic acid stains and either UV excitation of all three dyes or with a combination of UV excitation for the Hoechst 33342 dye and excitation by the 488 nm spectral line of the argon-ion laser. Differences in the intensity of the dye staining may make it difficult to simultaneously photograph the live, apoptotic and dead cells by microscopy. The kit components, number of assays and assay principles are summarized in Table 15.4.

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

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

Comet assays have traditionally been performed using ethidium bromide to stain the DNA. However, our YOYO-1 dye was found to increase the sensitivity of the assay eightfold compared to ethidium bromide. Use of the SYBR Gold and SYBR Green I stains 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 fragments in a template-independent fashion. Break sites have traditionally been labeled with biotinylated dUTP, followed by subsequent detection with an avidin or streptavidin conjugate (Section 7.6, Table 7.22). However, a more direct approach for detecting DNA strand breaks in apoptotic cells is possible via the use of our ChromaTide BODIPY FL-14-dUTP (C7614) as a TdT substrate ().

The single-step BODIPYFL dye–based assay has several advantages over indirect detection of biotinylated or haptenylated nucleotides, including fewer protocol steps and increased cell yields. BODIPY FL dye–labeled nucleotides have also proven superior to fluorescein-labeled nucleotides for detection of DNA strand breaks in apoptotic cells because they provide stronger signals, a narrower emission spectrum and less photobleaching (). Moreover, it has been reported that 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 (C7631) has been used similarly for a TdT-mediated apoptosis assay; presumably a number of the ChromaTide dUTP nucleotides listed in Table 8.7 could be used for the direct or indirect TUNEL assay; we have not yet tried the ChromaTide dCTP nucleotides in this assay. Furthermore, our anti-dye antibodies (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 amyotrophic lateral sclerosis. Analogous to TdT's ability to label double-strand breaks, the E. coli repair enzyme DNA polymerase I can be used to detect single-strand nicks, which appear as a relatively early step in some apoptotic processes. Because our ChromaTide BODIPY FL-14-dUTP (C7614) and ChromaTide fluorescein-12-dUTP (C7604) are incorporated into DNA by E. coli DNA polymerase I, it is likely that they may also be effective for in situ labeling with the nick translation method.

APO-BrdU TUNEL Assay Kit

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

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

* Terminal deoxynucleotidyl transferase (TdT), for catalyzing the addition of BrdUTP at the break sites

* 5-Bromo-2'-deoxyuridine 5'-triphosphate (BrdUTP)

* Alexa Fluor 488 dye–labeled anti-BrdU mouse monoclonal antibody PRB-1, for detecting BrdU labels

* Propidium iodide/RNase staining buffer, for quantitating total cellular DNA

* Reaction, wash and rinse buffers

* Positive control cells (a fixed human lymphoma cell line)

* Negative control cells (a fixed human lymphoma cell line)

* Detailed protocols (APO-BrdU TUNEL Assay Kit)

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

Apoptosis Assays Using Annexin V Conjugates

Annexin V Conjugates

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