彗星实验操作步骤(DNA损伤检测)

Lotte Bjergbæk (ed.), DNA Repair Protocols, Methods in Molecular Biology, vol. 920,DOI 10.1007/978-1-61779-998-3_6, © Springer Science+Business Media New York 2012C hapter 6T he Comet Assay: A Sensitive Genotoxicity Testfor the Detection of DNA Damage and RepairGünter S peit and A ndreas R othfussA bstractT he comet assay (single-cell gel electrophoresis) is a simple and sensitive method for studying DNA damage and repair. In this microgel electrophoresis technique, a small number of cells suspended in a thin agarose gel on a microscope slide is lysed, electrophoresed, and stained with a flu orescent DNA-binding dye. Cells with increased DNA damage display increased migration of chromosomal DNA from the nucleus towards the anode, which resembles the shape of a comet. The assay has manifold applications in fundamental research for DNA damage and repair, in genotoxicity testing of novel chemicals and pharmaceuticals, environmental biomonitoring, and human population monitoring. This chapter describes a standard protocol of the alkaline comet assay and points to some useful modi fic ations.K ey words:A lkaline comet assay ,A lkali-labile sites ,B iomonitoring ,C rosslinks ,D NA-strand breaks ,E xcision repair ,G enotoxicity testing ,S ingle-cell gel electrophoresisT he comet assay (single-cell gel electrophoresis) is a useful techniquefor studying DNA damage and repair with manifold applications.In this microgel electrophoresis technique, a small number of cellssuspended in a thin agarose gel on a microscope slide is lysed, electro-phoresed, and stained with a flu orescent DNA-binding dye. Cellswith increased DNA damage display increased migration of chro-mosomal DNA upon electrophoresis from the nucleus towards theanode, which resembles the shape of a comet (Fig. 1). In its alka-line version, which is mainly used, DNA-strand breaks and alkali-labile sites become apparent, and the extent of DNA migrationcorrelates with the amount of DNA damage in the cell. The cometassay combines the simplicity of biochemical techniques for detectingDNA single-strand breaks and/or alkali-labile sites with the singlecell approach typical of cytogenetic assays. The advantages of the 1.I ntroduction7980G. Speit and A. Rothfusscomet assay include its simple and rapid performance, its sensitivity for detecting DNA damage, the analysis of data at the level of the individual cell, the use of extremely small cell samples, and the usability of virtually any eukaryote cell population. Apart from image analysis, which greatly facilitates and enhances the possibili-ties of comet measurements, the cost of performing the assay is extremely low. The comet assay has already been used in many studies to assess DNA damage and repair induced by various agentsin a variety of cells in vitro and in vivo ( 1) . The test has widespread applications in genotoxicity testing in vitro and in vivo ( 2–5 ) , DNA damage and repair studies ( 6) , environmental biomonitoring ( 7,8 ) , human population monitoring ( 9, 10 ) , reproductive toxi-cology ( 11) , and radiobiology ( 12 ) .T he alkaline version (pH >13) of the comet assay introduced by Singh and coworkers ( 13) detects a broad spectrum of DNA lesions, that is, DNA single- and double-strand breaks and alkali-labile sites. Modi fi e d versions of the assay introduced by Olive ( 14) involved lysis in alkaline buffer followed by electrophoresis at either neutral or mild alkaline (pH 12.1) conditions to detect DNA double-strand breaks or single-strand breaks, respectively. However, since the majority of genotoxic agents induce much more single-strand breaks and alkali-labile sites than double-strand breaks, the alkaline version (pH >13) of the comet assay has been identi fi e d to show the highest sensitivity for detecting induced DNA damage and hasbeen recommended for genotoxicity testing (2 ) . Importantimprovements of the test procedure were introduced by Klaude F ig. 1. P hotomicrographs of human lymphocytes in the comet assay. ( a ) Untreated cell (control). ( b ) Cell exhibiting increased DNA migration after mutagen treatment.816 The Comet Assay: A Sensitive Genotoxicity Test for the Detection…and coworkers ( 15 ) . The use of agarose-precoated slides in combination with drying of gels and fi x ation of the comets led to a further simpli fi c ation and a much better handling of the test. The comet assay is especially suited for studies with a high number of samples since it can be performed in a high-throughput fashion and analysis of slides can be automated ( 16– 18 ) .A broad spectrum of DNA-damaging agents increases DNA migration in the comet assay such as ionizing radiation, hydrogenperoxide and other radical-forming chemicals, alkylating agents, polycyclic aromatic hydrocarbons (PHAs) and other adduct-forming chemicals, radiomimetic chemicals, various metals, or UV-irradiation. In principle, the alkaline version of the comet assay detects all kinds of directly induced DNA single-strand breaks and any lesion that can be transformed into a single-strand break under alkaline conditions (i.e., alkali-labile sites).I n addition to directly induced strand breakage, processes which introduce single-strand nicks in the DNA, such as incision during excision repair processes, are also detectable. In some cases (e.g., UV, PAHs) the contribution of excision repair to the induced DNA effects in the comet assay seems to be of major importance( 19) . Some speci fi c classes of DNA base damage can be detected with the comet assay in conjunction with lesion-speci fi c endonu-cleases ( 6) . These enzymes, applied to the slides for a short time after lysis, nick DNA at sites of speci fi c base alterations and the resulting single-strand breaks can be quanti fi e d in the comet assay. Using this modi fi c ation of the comet assay, oxidized DNA bases have been detected with high sensitivity with the help of endonu-clease III, formamidopyrimidine-DNA-glycosylase (FPG) or cell extracts in in vitro tests and in samples obtained from humanstudies( 6, 20– 22 ) .C rosslinks (DNA-DNA or DNA-protein) as induced by chemicals, such as nitrogen mustard, cisplatin, cyclophosphamide or formaldehyde may cause problems in the standard protocol. The induction of crosslinks reduces the ability of the DNA to migrate in the agarose gel by stabilizing chromosomal DNA( 23,24 ) . Crosslinks can be detected by adjusting the duration of unwinding and/or electrophoresis to such an extent that control cells exhibit signi fi c ant DNA migration. A lower extent in DNA migration in treated samples compared to untreated controls wouldthen indicate an induction of crosslinks ( 25) . Another possibility is to induce DNA migration with a second strand-breaking agent (e.g., ionizing radiation, methyl methanesulfonate) after exposure towards the (assumed) crosslinking agent and performing the comet assay immediately thereafter. A crosslinking effect is then determined as reduced migration in comparison with the effect ofthe strand-breaking agent alone ( 23,24 ) . Post-treatment of samples with Proteinase K allows to distinguish betweenDNA-DNA and DNA-protein cross-links ( 24) . 1.1.D etection of DNADamage82G. Speit and A. RothfussA widely used approach for determining DNA repair is to monitor a time-dependent removal of lesions (i.e., the decrease in DNAmigration) after treatment with a DNA-damaging agent. The comet assay has been successfully used to follow the rejoining of strand breaks induced by ionizing radiation or reactive oxygenspecies ( 26,27 ) as well as the repair of various kinds of DNA damage induced by chemical mutagens ( 28,29 ) . A useful extension of repair studies includes the additional use of lesion-speci fi cenzymes (6 ) , or cell extracts ( 30, 31 ) . Thereby, the repair of speci fi c types of DNA lesions can be followed and, due to its high sensitiv-ity, this approach enables the analysis of very low (“physiological”)levels of DNA damage ( 32) . A common alternative approach is the use of repair inhibitors or repair-de fi c ient cells. Incubation of cells with inhibitors of DNA- (repair-) synthesis, such as hydroxyurea, cytosine arabinoside, or aphidicolin leads to an accumulation ofincomplete repair sites as DNA breaks ( 19,33 ) . Mutant cell lines either with a speci fi c defect in a repair pathway (e.g., xeroderma pigmentosum) or with a hypersensitivity towards speci fi c DNA damaging agents (e.g., various mutant rodent cell lines) are well suited to elucidate DNA repair pathways and the biological conse-quences of disturbed DNA repair or to evaluate the repair compe-tence of cells ( 19,34– 36 ) . While the standard version of the comet assay provides information on DNA damage and repair in the whole genome of a cell, the introduction of a combination of the comet assay with fl u orescence in situ hybridization (FISH) addition-ally allows to measure DNA damage and repair in speci fi c genomicregions ( 37–39 ) .T he purpose of this protocol is to provide information on the application of the alkaline comet assay for the investigation of DNA damage and repair in mammalian cells in vitro. For establishing the method, we recommend to start with experiments using blood samples and the induction of DNA damage by a standard mutagen (e.g., methyl methane sulfonate, MMS). The method described here is based on a protocol established by R. Tice according to theoriginal work of Singh et al. (13 ) and includes the modi fi c ations introduced by Klaude and coworkers ( 15) . An outline of the pro-tocol is diagrammed in Fig.2 .1. M icroscope slides (with frosted end).2.C overslips (24 × 60 mm). 3. N ormal melting-point agarose.4. L ow melting point (LMP) agarose.5. H orizontal gel electrophoresis unit.1.2.M easuring DNA Repair 2.M aterials836 The Comet Assay: A Sensitive Genotoxicity Test for the Detection… 6. F luorescence microscope equipped with an excitation fi l ter of515–560 nm and a barrier fi l ter of 590 nm.7.P hosphate-buffered saline (PBS) (without Ca 2+ and Mg 2+ ). 8.L ysing solution (1 L): 2.5 M NaCl, 100 mM EDTA, 10 mM Tris (set pH to 10.0 with ~7 g solid NaOH). Store at room temperature. Final lysing solution (100 mL, made fresh): add 1 mL of Triton X-100 and 10 mL of DMSO to 89 mL of lysingsolution, and then refrigerate (4 °C) for 60 min before use.9.E lectrophoresis buffer: 300 mM NaOH/1 mM EDTA. Prepare from stock solutions of 10 N NaOH (200 g/500 mL distilled H 2 O ), 200 mM EDTA (14.89 g/200 mL of dH 2 O , pH 10.0). Store at room temperature. For 1× buffer, mix 45 mL NaOH,7.5 mL of EDTA, and add water to 1,500 mL (total volume needed depends on gel box capacity). Mix well and store at 4 °C. Make fresh before each run.10.N eutralization buffer: 0.4 M Tris–HCl, pH 7.5. Store at room temperature.11. E thidium bromide staining solution: 10× stock: 200 μ g /mL.Store at room temperature. For 1× stock (20 μ g /mL), mix1 mL with 9 mL dH2 O and fi l ter. C aution : Ethidium bromide is a mutagen. Handle with care.StainingNeutralisationF ig.2.S cheme for the performance of the comet assay.84G. Speit and A. Rothfuss1. C lean slides with ethanol before use. Wear gloves.2. S cratch slides with a diamond pen, drawing a line width-wise approximately 5 mm from the end of the slide to improve the adhesion of the agarose.3. F or the bottom layer, prepare 1.5 % normal melting agarose (300 mg in 20 mL of PBS) and boil until the agarose is com-pletely melted. Dip the slides brie fl y into hot (>60 °C) agarose. The agarose should reach to and cover half of the frosted part of the slide to ensure that the agarose will stick properly. Wipe off the agarose from the bottom side of the slide and lay the slide horizontally. This step has to be performed quickly to ensure a good distribution of agarose. Dry slides overnight at room temperature. Slides can be stored for several weeks.4. P repare 0.5 % LMP agarose (100 mg in 20 mL of PBS). Microwave or heat until near boiling and the agarose dissolves. Place the LMP agarose in a 37 °C water bath to cool.5. A dd 120 μ L of LMP agarose (37 °C) mixed with 5,000–50,000 cells (see Subheading 3.2 ) in ~5–10 μ L (do not use more than 10 μ L ). Add coverslip, and place the slide in a refrigerator for ~2 min (until the agarose layer hardens). Using ~10,000 cells results in ~1 cell per microscope fi e ld (250× magni fi c ation). From this step until the end of electrophoresis, direct light irradiation should be avoided to prevent additional DNA damage.6. G ently slip off the coverslip and slowly lower slide into cold, freshly made lysing solution. Protect from light, and place at 4 °C for a minimum of 1 h. Slides may be stored for extended periods of time in cold lysing solution (but generally not lon-ger than 4 week). If precipitation of the lysing solution is observed, slides should be rinsed carefully with distilled water before electrophoresis. 1. W hole blood: Mix ~5 μ L whole blood with 120 μ L of LMP agarose, and layer onto the slide.2. I solated lymphocytes: Add 4 mL of whole blood to a tube with4 mL prewarmed (37 °C) Ficoll. Centrifuge for 25 min at ~320 × g . Carefully remove the lymphocytes and resuspend them in 8 mL RPMI 1640 medium. Centrifuge again for 10 min at ~180 × g . Remove the supernatant and repeat the washing step. Incubate the cells for 30 min at 37 °C. Centrifugefor 10 min at ~130 × g , discard the supernatant and resuspend the pellet in 375 μ L of RPMI 1640 medium. Count the cells and adjust to 1,500 cells/ μ L . Mix 10 μ L of the suspension with 120 μ L LMP agarose and layer onto the slide.3.M ethods (S eeN otes 1and 2)3.1.P reparationof Slides3.2. P reparation of Cells( S ee N otes 3 and 4)856 The Comet Assay: A Sensitive Genotoxicity Test for the Detection… 3. C ell cultures. (a) M onolayer cultures: Gently trypsinize the cells (for approx. 2 min with 0.15 % trypsin, stop by adding serum or com-plete cell culture medium) to yield approximately 1 × 10 6 cells/mL. Add 10 μ L of cell suspension to 120 μ L LMP agarose, and layer onto the slide. (b) S uspension cultures: Add ~15,000 cells in 10 μ L (or smaller volume) to 120 μ L of LMP agarose and layer onto the slide. 1. A fter at least 1 h at 4 °C, gently remove the slides from the lysing solution. 2. P lace the slides in the gel box near the anode (+) end, posi-tioning them as close together as possible. 3. F ill the buffer reservoirs with electrophoresis buffer (4 °C) until the slides are completely covered (avoid bubbles over the agarose). Perform the electrophoresis in an ice bath (4 °C). 4. L et slides sit in the alkaline buffer for 20–60 min to allow unwinding of the DNA and the expression of alkali-labile damage. For most experiments with cultured cells, 20 min are recommended. 5. T urn on power supply to 25 V (~0.8–1.5 V/cm, depending on gel box size) and adjust current to 300 mA by slowly raising or lowering the buffer level. Depending on the purpose of the study and on the extent of migration in control samples, allow the electrophoresis to run for 20–40 min. For most experi-ments, 20 min is recommended. 6. T urn off the power. Gently lift the slides from the buffer and place on a staining tray. Coat the slides with drops of neutralization buffer, and let sit for at least 5 min. Repeat two more times. 7. D rain the slides, rinse carefully with distilled water, and let them dry (inclined) at room temperature. Slides can be stored for a longer time before staining. To stain, rinse the slides brie fl y in distilled water, add 30 μ L 1× ethidium bromide staining solution, and cover with a coverslip. Antifade can be used to prevent slides from drying or fading out if necessary, i.e., when automated analysis is used. S lides should be stained one by one and evaluated immedi-ately. It is possible to rinse stained (evaluated) slides in distilled water, remove the coverslip, let the slides dry and stain them at a later time point for reevaluation. F or visualization of DNA damage, observations are made of ethid-ium bromide-stained DNA at 250× (or 400×) magni fi c ation usinga fl u orescence microscope. Generally, 100 randomly selected cells3.3.E lectrophoresisand Staining ( S eeN otes 5–7)3.4.E valuation of DNAEffects (S ee N ote 8)86G. Speit and A. Rothfussper sample are analyzed. In principle, evaluation can be done infour different ways:1. I mage analysis systems are used to quantify DNA damage.Parameters such as tail intensity (percentage DNA in the tail),tail moment, and tail length are commonly used. It is impor-tant to note that the some parameters (e.g., tail moment) maybe calculated differently among image analysis systems. For thepurpose of inter-laboratory comparison of DNA damageparameters, tail intensity is probably the most suited.2. A utomated systems have been established, which search for com-ets and carry out the analysis with minimal human intervention.3. C ells are scored visually according to tail size into fiv e classes(from undamaged, 0, to maximally damaged, 4). Thus thetotal score for 100 comets can range from 0 (all undamaged)to 400 (all maximally damaged).4. C ells are analyzed using a calibrated scale in the ocular lens ofthe microscope. For each cell, the image length (diameter of thenucleus plus migrated DNA) is measured in microns, and themean is calculated. Alternatively, the length of the comet (orcomet tail) can be measured on a photomicrograph. These mea-surements are very laborious and may only give limited informa-tion because the tail length saturates at higher levels of damage.F or the statistical analysis of comet assay data, a variety of para-metric and nonparametric statistical methods are used. The mostappropriate means of statistical analysis depends on the kind ofstudy and has to take into account the various sources of assay vari-ability. For a powerful statistical analysis of in vitro test data, appro-priate replication and repeat experiments have to be performed (2,40, 41). For example, the median DNA migration of 50 cells persample and the mean of 2–3 samples per data point may be deter-mined. Also, the mean from repeat experiments can be determined.The use of the median should be preferred over the average since anormal size distribution is usually not observed. Analyses are mainlybased on changes in group mean response but attention shouldalso be paid to the distribution among cells which often providesadditional important information. Recommendations for appropriatestatistical analyses of comet assay data have been published (40, 41).4.N otes1. M any technical variables have been used including the concen-tration and amount of LMP agarose, the composition of thelysing solution and the lysis time, the alkaline unwinding, theelectrophoresis buffer, electrophoretic conditions, and6 The Comet Assay: A Sensitive Genotoxicity Test for the Detection…87DNA-speci fic dyes for staining. Some of these variables mayaffect the sensitivity of the test. To allow for a comparisonobtained in different laboratories and for a critical evaluationof data, it is absolutely necessary to clearly describe the tech-nical details of the method employed.2. T he simplicity of the comet assay combined with the need ofonly low number of cells per sample enables the conduct ofin vitro studies with high ef fic iency. Therefore, the comet assaycan be used in a high-throughput fashion (17). Furthermore,the introduction of automated image analysis systems forcomet assay slides further can speed up test performance (18).3. M any other cell types have been used and it is an advantage ofthe comet assay that virtually any eukaryote cell population isamenable to analysis. The comet assay is particularly suited forthe investigation of organ- or tissue-speci fic genotoxic effectsin vivo (2–5), the only requirement being the preparation ofan intact single cell suspension.4. F or the demonstration of a positive effect, mix 200 μLheparinized whole blood with 50 μL of a 2.5 × 10 −4M methylmethanesulfonate (MMS) solution ( fin al concentration:5 × 10 −5M), incubate for 1 h at 37 °C and then use 10 μL forthe test.5. F or each cell type, the method should be adjusted scienti fic allyto obtain valid and reproducible results. It is important to de fin ethe optimal time for alkaline treatment and electrophoresis. It isrecommended that the conditions must be such that the DNAfrom the control cells exhibit, on the average, some migration.This effect ensures sensitivity and enables an evaluation ofintralaboratory experiment-to-experiment variability (5).6. T he temperature during alkaline treatment and electrophoresissigni fic antly in flu ences the amount of DNA migration (42). It isnecessary to establish stable and reproducible conditions and itmay be useful to use a cooled electrophoresis unit or to place theelectrophoresis unit in a jar fil led with ice or in a cooled room.7. I f speci fic types of base damage should be determined by usinglesion-speci fic endonucleases or cell extracts, the standard proto-col has to be modi fie d in the following way: after at least 1 h at4 °C, gently remove slides from the lysing solution and washthree times in enzyme buffer. Drain slides and cover with 200 μLof either buffer or enzyme in buffer. Seal with a coverslip andincubate for 30 min at 37 °C. Remove coverslip, rinse slides withPBS and place them on the electrophoretic box (6, 20–22).8. I t is strongly recommended to include some measure of cyto-toxicity into any study since increased DNA migration may alsooccur due to non-genotoxic cell killing. However, such an effectmay depend on the cell type used. While no increased DNAmigration had been observed in human leukocytes (43)or cell88G. Speit and A. Rothfusslines such as V79 (43, 44)and L5178Y (45), TK-6 cells showedincreased DNA migration after treatment with non-genotoxiccytotoxins when viability in treated cultures fell below 75 % (46).Therefore, acute cytotoxic effects should be determined byTrypan blue exclusion measurements or flu orochrome-mediatedviability tests. Furthermore, individual dead or dying cells maybe identi fie d by their speci fic microscopical image, i.e., necroticor apoptotic cells may result in comets with small or nonexis-tent head and large, diffuse tails (47). 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