Methods in Apoptosis
Apoptosis and necrosis are two mechanisms of cell death, each with its own distinguishing morphological and biochemical features. Necrosis, which occurs within seconds of cell insult (Majno and Joris, 1995), may be described as "cell murder" resulting from external damage to the cell membrane, loss of homeostasis, water and extracellular ion influx, intracellular organelle swelling, cell rupture (lysis), and so inflammatory cell attraction. Initially described by Kerr et al. (1972), apoptosis is a much slower process of events than necrosis, requiring from a few hours to several days (depending on the initiator) and resulting from molecular signals initiated within individual cells (see Nagata, 1997; Barinaga, 1998; Van Cruchten and Van Den Broeck, 2002). The initiators of apoptosis that instigate the cascade of events leading to activation of a series of cytoplasmic proteases, termed caspases (cysteinyl-asparatate-specific proteinases), are multiple. Two such pathways involve (i) activation of cell surface death receptors, resulting in direct activation of caspases, and (ii) cytochrome c release from the mitochondria into the cytoplasm following induction of leakiness in its membrane. The terminal caspases downstream from these initiator mechanisms lead to the morphological and biochemical events of apoptosis.
The mechanisms of apoptosis, which is analogous to "cell suicide," are essentially the same, whether induced by genetic signals or through external initiators. The events involved include cell membrane blebbing; chromatin aggregation; nuclear and cytoplasmic condensation leading to cell shrinkage; and partitioning of cytoplasm and nucleus into membrane-bound apoptotic bodies, which contain ribosomes, morphologically intact mitochondria, and nuclear material. As a result of the efficient mechanism for the removal of apoptotic cells by phagocytic cells in vivo, an inflammatory response is not stimulated. In vitro apoptotic bodies swell and finally lyse (Darzynkiewicz and Traganos, 1998; Kiechle and Zhang, 2002).
Apoptosis is key to many fundamental aspects of biology, including embryonic development and normal tissue homeostasis, as well as in many pathological events, such as loss of regulated cell death in cancer, response of cancer cells to chemo- and radiotherapy (Clynes et al., 1998), and death of cells in diabetes (Sesti, 2002) and neurodegenerative diseases (Vila and Przedborski, 2003). Accurate detection of apoptosis is of great importance to increase our understanding of biological events that may allow us to understand and to manipulate these events as a form of therapy.
Major elements involved in the apoptotic pathway, which should be considered when selecting suitable methods for apoptosis detection, include the following.
The range of techniques and methods for analysis of apoptosis is extensive. Due to space limitations in this article, we do not propose to describe all methods comprehensively. Table I lists a number of techniques for analysis of the cellular events described earlier. A selection of techniques used for studying these events is described in further detail.
II. MATERIALS AND INSTRUMENTATION
A. Light Microscopy (LM) and Fluorescence Microscopy (FM)
Frost-ended slides and coverslips (Chance Propper); ice-cold methanol; Coplin jars; forceps; micropipettes; grease pen (DAKO S2002); and mounting medium (Vectashield mounting solution with antifade additive [Vector Labs.; H-1000]) suitable for fluorescence slides and may also be used for LM slides. Alternatively, 20% glycerol prepared in H2O is also suitable for mounting slides for LM.
If analysing suspension cells, a cytospin (e.g., Heraeus Labofuge 400) and cytospin cups are required.
For LM only: haematoxylin, aluminium potassium sulphate, citric acid, and chloral hydrate.
For FM only: Stains include 4',6-diamidino-2- phenylindole (DAPI, Sigma D-9542), propidium iodide (PI, Sigma P-4170), Hoechst 33258 (Sigma B-2883), Hoechst 33342 (Sigma B-2261), and acridine orange (AO, Sigma A-6014) in phosphate-buffered saline (PBS), pH 7.4.
B. Gel Electrophoresis for DNA Ladder
Horizontal agarose gel electrophoresis chamber and combs (Bio-Rad); electric power supply; UV transilluminator or gel analyser (e.g., EpiChemi II Darkroom, UVP Laboratory Products); PBS (Oxoid BR14a); ethidium bromide (Sigma E-8751); agarose (Sigma A-9539); Tris (Sigma T-8524); EDTA (Tris E-5134); NaCl (Sigma S-9899); sodium dodecylsulfate (SDS) (BDH 442152V); RNase A (Sigma R-5250); proteinase K (Sigma P-2308); boric acid (Sigma B-7901); bromphenol blue (Sigma B- 5525); glycerol (Sigma G-2025); molecular markers, e.g., Phi X174 DNA HaeIII digest (Sigma D-0672); micropipettes.
C. Terminal Deoxynucleotidyl Transferase- Mediated Deoxyuridine Triphosphate Nick End-Labelling Assay (TUNEL)
Apoptosis detection system: (1) fluoresceincontaining equilibrating buffer, (2) nucleotide mix, (3) TdT enzyme, (4) 20X SSC solution, (5) proteinase K, (6) protocol (Promega; G3250); plastic coverslips, glass slides and coverslips (Chance Propper); propidium iodide (Sigma P-4170); Coplin jars; forceps; humidifying chamber; 37°C incubator; Triton X-100 (Sigma T- 8787), PBS (Oxoid BR14a); 4% paraformaldehyde (Sigma P-6148) in PBS (pH 7.4) (freshly prepared); Vectashield mounting solution with antifade additive (Vector Labs.; H-1000); 70% ethanol [prepare from absolute ethanol (Sigma E-7037)]; and micropipettes.
Note: Items 1-6 are included in the apoptosis detection system available commercially from Promega (G3250). There are, however, other detection kits available commercially that may be equally suitable.
D. Reverse Transcriptase-Polymerase Chain Reaction
As all general laboratory glassware, spatulas, etc., are often contaminated by RNases, these items should be treated by baking at 180~ for a minimum of 8h. Sterile, disposable plasticware is essentially free from RNases and so generally does not require pretreatment. All solutions/buffers used should be prepared in baked glassware using sterile ultrapure water treated by the addition of diethylpyrocarbonate (DEPC) [Sigma D-5758, (0.1%, v/v)] and autoclaved. As for all laboratory procedures described in this article, gloves should be worn at all times to protect both the operator and the experiment. This, too, prevents the introduction of RNases and foreign RNA or DNA in the reverse transcriptase (RT) and polymerase chain reaction (PCR).
1. For RNA Isolation and Quantification
TRI Reagent (Sigma T-9424), chloroform (Sigma C-2432), isopropanol (Sigma 1-9516), ethanol [Sigma E-7037; prepare as 75% (v/v) in H2O], DEPC, micropipettors, tips, Eppendorf tubes, etc., spectrophotometer (e.g., SpectraMax Plus plate reader, Molecular Devices), and quartz cuvettes or Nanodrop (ND-1000; Labtech Int. Ltd.)
2. For RT and PCR Reactions
DEPC-treated H2O; oligo(dT)12-18mer (Oswel, Southampton, UK); MMLV-RT enzyme (200U/µl) (Sigma M-1302); 5X buffer (Sigma B-0175); dithiothreitol (DTT, 100mM) (Sigma D-6059); RNasin (40U/ml) (Sigma R-2520); dNTPs (10mM each of dATP, dCTP, dGTP, and dTTP for RT; 1.25 mM each for PCR) (Sigma DNTP-100); MgCl2 (25 mM) (Sigma M-8787), Taq DNA polymerase enzyme (5U/µl) (Sigma D-6677); 10X buffer (Sigma P-2317); target primers and internal control primer (see Table II) (for further details on primer selection criteria, see O'Driscoll et al., 1993); thermocycler.
3. Gel Electrophoresis
Amplified products are analysed by gel electrophoresis (see Section IIIB).
Note: Several of the stains and other reagents used (e.g., DEPC) should be handled with caution as they are known or thought to be toxic, carcinogenic, and/or mutagenic.
A. Light and Fluorescence Microscopy Detection of Apoptotic Cell Morphology
As described previously, a cell undergoing apoptosis proceeds through various stages of morphological change (Wilson and Potten, 1999). Light microscopy and fluorescence microscopy are probably the simplest and most basic techniques by which such apoptotic cell death can be investigated. A broad range of stains and dyes are available to assist in the assessment of nuclear morphology. For light microscopy, the nuclear stain haematoxylin is used frequently (often with eosin as a counterstain). The most commonly used DNA nucleic acid-reactive fluorochromes for UV light fluorescence microscopic analysis of fixed (porated using, e.g., methanol or ethanol) cells include DAPI, PI, Hoechst 33258 and 33342, and AO. (As mentioned previously, PI, DAPI, and Hoechst are also very useful for assessing the membrane permeability of cells).
Using LM, cells dying by apoptosis are identified by their reduced size, cell membrane "blebbing/ budding," and loss of normal nuclear structural features- nuclear fragmentation and chromatin condensation. In contrast, characteristic features of necrotic cell death include cell and nuclear swelling, cytoplasmic vacuolisation, patchy chromatin condensation, and plasma membrane rupture.
If assessing adherent cell types, the cells may be grown directly on coverslips (plated in Petri dishes). For suspension cells, cytospins are generally prepared. For both monolayer and suspension cultures, it is important to consider cell concentration. Fifty to 70% confluency of the area being analysed is generally considered optimalmif the cells are more confluent, overlapping may occur, which may hinder analysis; if cells are too sparse, an accurate examination may not be achievable. Typically, 1-2 × 105 cells suspended in 100µl PBS containing 1% (w/v) fetal calf serum (FCS) should be cytocentrifuged onto microscope slides using a low-speed, short centrifugation (e.g., 600- 1000rpm for 2-4min); however, this should be optimised as relevant for cells being analysed.
For LM only. 0.1M haematoxylin: In a fume hood, dissolve 1 g haematoxylin (BDH 34242) in 1 litre distilled water, boil for 5 min, remove from heat, and add 0.2 g sodium iodate (Sigma S-4007). After 10 min, in the order listed, add 50g aluminium potassium sulphate (Sigma A-7167), 1 g citric acid (Sigma C-2404), and 50g chloral hydrate (Sigma C-8383), allowing each to dissolve completely prior to adding the next. The complete solution is stable for approximately 3 months at room temperature.
For FM only. Fluorescent stains should be stored in light-proof containers to prevent quenching.
Note: When working with fluorochromes, minimise exposure to light at all stages of preparation.
B. Gel Electrophoresis for DNA Ladder
Activation of endogenous Ca2+- and Mg2+- dependent nuclear endonuclease(s) cleaves DNA into discrete fragments, initially into 300- to 50-kb fragments and subsequently into 180-bp fragments. In brief, for DNA fragment detection by gel electrophoresis, DNA is extracted from cells and loaded onto a 1.5% agarose gel containing ethidium bromide. The DNA fragments form a characteristic "ladder" pattern resulting from multiples of a 180-bp DNA subunit, representing DNA of the size of individual nucleosomes and oligonucleosomes. [Nuclear DNA damage resulting in death by necrosis, however, is random and results in smears on a gel (Ramachandra and Studzinski, 1995).]
TUNEL is a cytochemical method suitable for analysis of apoptotic DNA fragmentation in individual cells. This technique involves in situ enzymatic labelling of the 3'-OH ends of fragmented DNA in fixed, permeabilised cells with either enzyme (phosphatase or peroxidase) or fluorochrome-tagged deoxynucleotides using terminal deoxynucleotidyl transferase (TdT). (Note: If DNA polymerase I is used instead of TdT, the method is termed in situ nick translation.) If fluorescence labelling is chosen, the fluorescein-12-dUTP-labelled DNA can then be visualised directly by fluorescence microscopy to give qualitative results. If facilities are available and quantitative results are required, flow cytometric analysis may be used.
Expression of apoptosis-related mRNAs may be analysed using RT-PCR methods. This section describes the use of "basic" RT-PCR, but it is important to be aware that depending on the requirements of the study and the resources available, RT-PCR may be used to indicate the presence or absence of transcripts of interest or it may be developed as a semiquantitative or a quantitative level using real-time PCR.
RNA may be isolated from cells using the procedure described by Chomczynski and Sacchi (1987). However, there are now a number of less laborious, commercially available methods, including the use of TRI reagent (Sigma), as described here. The RT procedure detailed involves use of the MMLV-RT (Sigma) enzyme for cDNA synthesis and the PCR uses Taq DNA polymerase (Sigma). Again, however, there are other reverse transcriptases and DNA polymerase enzymes available that may be equally suitable.
Primers suitable for the analysis of human bcl-2, bag-l, bax-α, mcl-1, galectin-3 (designed in our laboratory), and survivin are included in Table II, as examples of apoptosis-related gene transcripts that may be desirable to analyse by RT-PCR. There are, of course, many more mRNAs involved in apoptotic pathways that should be considered. The methods described here may be adapted by using relevant primers for the amplification of other apoptosis-related cDNAs [for information on primer design and selection criteria, see O'Driscoll et al. (1993)].
Table II also includes primer sequences for coamplification of cDNA derived from an endogenous "housekeeping" mRNA, β-actin, as control. Inclusion of such primers serves to indicate that the RT and PCR reactions have been performed successfully; if performing semiquantitative PCR, this allows the apoptosis-related gene transcript results to be normalised relative to a control that (generally) should be constant.
RNA Isolation and Quantitation
Typical RT Reaction
Typical PCR Reaction
IV. SELECTION OF A SUITABLE METHOD FOR APOPTOSIS DETECTION
There are many factors to be considered when selecting a suitable method for investigating apoptosis. These factors include the cell type being analysed, the nature of the cell death inducer, the stage of cell death, the information required from the study (e.g., whether information on single cells or on the cell population, as a whole, is required), and the resources available (e.g., where or not access is available to a fluorescence microscope, flow/cytometer, thermocycler, etc.). To assist with selection, Table III summarises the "advantages" and "disadvantages" of the procedures detailed in this article. To form a more extensive understanding of the events occurring within cells, it is advisable, whenever possible, to investigate cell death using more than one technique.
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