Flash-comet assay

(1)

ContentslistsavailableatScienceDirect

MethodsX

journal homepage:www.elsevier.com/locate/mex

MethodArticle

Flash-comet assay

Erik Bivehed^∗,Björn Hellman

Uppsala University, Department of Pharmaceutical Biosciences, Drug Safety and Toxicology, Uppsala, Sweden

abstract

In the present paper, we present a substantially revised protocol ofthe widely used SCGE assay performed under alkaline conditions.In our updated version ofthe comet assay, whichwe call the Flash-comet, LiOH is used insteadof NaOH during the unwindingand electrophoresis. Thisallows ahigher voltage during the electrophoresis(5V/cm insteadof0.7V/cm),makingitpossibletoreducetheunwindingtimefrom20 to40 to2.5min,andtheelectrophoresistimefrom10to20to1min.Still,theFlash-cometwasfoundtodetectDNA strand breaksandalkali-labile siteswithahigherdegreeofsensitivitythantheconventionalprotocolincells thathad beenexposedtoH2O2 orionizingradiation.InordertopreventalkalinehydrolysisofDNA,thewash andlysissolutionshavebeenmodiﬁedintheFlash-cometprotocol.

• ByusinganalkalineLiOH-basedmedium,theFlash-cometallowsformuchshortertimesforbothunwinding andelectrophoresisthantheconventionalcometassaywithoutcompromisingthesensitivity.

• Thereducedrun-timesoftheunwindingandelectrophoresisstepsintheFlash-cometshouldalsoreducethe riskoflaboratory-inducedalkalinehydrolysisofDNAwhenevaluatingthepotentialDNA-damagingeffectsof differenttypesofxenobiotics.

ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/)

article info Method name: Flash-comet

Keywords: DNA-damage, Gel electrophoresis, Genotoxicity testing, Gentle lysis, In vitro comet assay, Low conductivity electrophoresis solution, Single cell analysis

Article history: Received 25 August 2020; Accepted 21 November 2020; Available online 27 November 2020

DOI of original article: 10.1016/j.mrgentox.2020.503240

∗ Corresponding author.

E-mail address: erik.bivehed@farmbio.uu.se (E. Bivehed).

https://doi.org/10.1016/j.mex.2020.101161

(2)

SpeciﬁcationsTable

Subject Area: Pharmacology, Toxicology and Pharmaceutical Science More speciﬁc subject area: Genetic toxicology

Method name: Flash-comet

Name and reference of original method:

Alkaline single cell gel electrophoresis assay (SCGE)/Alkaline comet assay (1,2) Resource availability:

Methoddetails

ThegeneralstructureofFlash-cometassayissimilar tothatofthesinglecellgelelectrophoresis assay,originallypresentedbyÖstlingandJohansonandperformedunderneutralconditions[1].The originalprotocolwaslatermodifiedbySinghetal.whoperformedtheassayunderalkalineconditions [2]. In comet assay (conventional and Flash), the cells are exposed to xenobiotics when the cells are dispersed in medium. After exposure, the cellsare embeddedin low-melting point agarose on microscopeslidesoronGelBond^R films.Theslides/filmsarethensubmergedinacoldlysissolution.

This solution containsdetergents andhighly concentrated saltsolution in order to disruptcellular membranes and proteins leaving a protein depleted nucleoid. The samples are then subjected to alkaliunwindinginanelectrophoresissolution(pH>13)inordertogeneraterelaxationofthesuper- coiled DNAstructure to releaseDNA-loops andfragments [2-5]. After unwinding, electrophoresis is performed by applying a strong electric field which will cause migration of the fragmented and relaxednegatively-chargedDNAtowardstheanode[6].TheamountofDNAwhichmigratestowards the anode will depend on the severity of the DNA-damage in the cell. After electrophoresis and staining, a cell with significant DNA-damage (DNA strand breaks) will look like a comet in the microscope. The level ofDNA-damage is usually evaluated by measuring the fluorescence intensity ofthecometusingimagingsoftware(CometassayIV;Perepectiveinstruments,UK).Thereareseveral parameters thatcouldbe used,butthemostcommonisprobablythe percentageofDNAinthetail (%TDNA).

Inthepresentpaper,wedescribearatherextensivelymodiﬁedprotocolofthealkalineversionof thecometassay,whichwecalltheFlash-cometassay.Themajoralterationoftheprotocolcompared toconventionalone,istheintroductionofalow-conductiveelectrophoresissolution(30mMLiOH;pH 12.5),allowingtheelectrophoresistobeperformedatsix-timeshighervoltage.Theincreasedvoltage results in an increasedsensitivity ofthe assay andalso reducesthe electrophoresis run-time from 10to 20minto1min.Furthermore,duetothehighvoltageused,theunwindingtime canalsobe reducedfrom20to40mindownto2.5min.Thecompositionofthewashandlysissolutionsisalso modiﬁedin theFlash-comet, mainly by loweringthe pH from10 to8.5 inorder toprevent assay- inducedalkalinehydrolysisandDNAdenaturation.

Methodprocedure Equipmentandconsumables

- Analyticalbalance - pHmeter

- Centrifuge(preferablewithcooling) - Microwaveoven

- Magneticstirrer

- Horizontalsubmarinegelelectrophoresistank (largesize;gelplatformL× W:20× 20cm;Buffer volume:>2000ml)

- Adjustablepowersupply - Glassbeakers

- Erlenmeyerﬂasks - Measuringglass

(3)

- Stainingglasscuvettes - Slidetray

- Stainingbox(ambercolor) - Glassslides(76× 26mm) - Coverslips(24× 60mm) - Coverslips(50× 60mm) - GelBond^R ﬁlm

- Bluntforceps - Micropipettes

- Eppendorftubes(1.5mland5ml) - Slidestoragebox

- Fluorescencemicroscope

- Charge-coupleddevice(CCD)camera

- Analysissoftware– CometAssayIV(PerspectiveInstruments,UK)orsimilar Stock,ready-to-useandworkingsolutions

The only solutions that are boughtasready-to-use are the cell growthmedia, supplementsand antibiotic forcellculture(requirmentsvarybetweencelltypea)andphosphate-bufferedsaline(PBS).

Allother solutionsmustbepreparedormodiﬁedin-house.Someofthemcanbestoredformonths, eitheratroomtemperature(RT)orat4°C.Theworkingsolutionsmustbepreparedonthesameday theassayisperformed.

Washsolution,1000ml,pH8.5:(1mMKH₂PO₄,155mMNaClNa₂HPO₄–7H₂O,adjustedwithNaOHto pH8.5)

- Mg-andCa-freePBS - AdjustpHto8.5usingNaOH

- Storeat4°C(canbestoredformonthsunderthiscondition)

Lysissolution,pH8.5

- NaCl(pH8.5)5M(canbestoredatRTformonths) - EDTA(pH8.5)0.25M(canbestoredatRTformonths) - Trisbase(pH8.5)250mM(canbestoredatRTformonths) - TritonX-100orequivalentdetergent

- DMSO

Lysisworkingsolution,150ml:(2.5MNaCl,100mMNa₂-EDTA,10mMTris,1%TritonX-100,5%DMSO andadjustedwithNaOHtopH8.5)

- 75mlNaCl,5M,pH8.5 - 60mlEDTA,0.25M,pH8.5 - 7.5mlDMSO

- 1.5mlTritonX-100(orequivalentdetergent)

- 6.0ml250mMTrisadjustedtopHto8.5usingNaOH - Cooldownto4°C

Electrophoresissolution,pH12.8

- LiOH(pH12.8)3–4M(canbestoredatRTformonths)

Electrophoresisworkingsolution,2000ml,pH12.5:(30mMLiOH,pH12.5) - 15ml4MLiOH

- Diluteto2000mlusingdeionizedH2O - Cooldownto4°C

(4)

Neutralizationbuffer,1000ml,pH7.5:(0.4MTris-base,adjustedwithHCltopH7.5)

- Tris-base0.4MinH₂O - AdjustpHto7.5usingHCl - DilutewithH₂Oto1000ml

- Storeat4°C(canbestoredformonths) Slidepreparation

This isnot an absolute requirementandcan be omitted ormodiﬁed dependingon thelab set- up andpreference.However, thispartofthe assaycan preferablybe conductedinadvanceinlarge batches. In orderto minimize bias, we recommend to assign a numberor letterto each slide and randomly assign them to treatment groups. For example, a full experiment with seven treatment groupsrequires21slides(i.e.threeslidespergroup).TheuseofGelBond^R isnotnecessaryalthough ithighlyimprovesgelstabilityandassuresﬁrmadherence.Oncepreparedtheslidesarebeststored inadryanddust-freeenvironment.

1. CutsheetsofGelBond^R (Lonza,Switzerland)into25× 55mmstrips.

2. AddasmallamountoftheglueNorlandOpticalAdhesive68(ThorlabsInc.,US-NJ)totheedge ofthefrostedpartofamicroscopeslide(26× 76mm)

3. PlacetheGelbond^R striponthemicroscopeslidewiththehydrophobicsidedown(waterwill beaduponthehydrophobicside)

4. ApplylightpressuretoensurethatbothGelbond^R andmicroscopeslideareconnectedviathe glue

5. IrradiatetheslidewithUV-light(365nm)for20s.

6. Repeatsteps2–5foralloftheslides.

PBS-agaroseforsinglecellentrapment

Theagaroseisdissolved inMg-andCa Mg-andCa-freePBS(pH7.4),inordertomaintainionic pressure andto prevent triggering premature cell death. The stock solution can be aliquoted and storedat4°Cforseveralmonths.

1. InasmallErlenmeyerﬂask, addlow-meltingpointagarosewithMg-andCa-freePBS(pH7.4) toaﬁnalconcentrationof0.6%(w/w).Makesuretorecordthetotalweight.

2. Heat the mixture in a microwave (150 W for 3 min, avoid boiling) until the agarose has dissolvedcompletely.

3. Let the solution cool down to 37°C. Weigh the ﬂask again and add deionized H₂O for adjustmenttotheoriginalweight.

Pre-assaypreparations

1. Pre-heatwaterbathandheatplateto37°C 2. Startwarmingcellgrowthmedium

3. Preparelysisandelectrophoresisworkingsolutionsandcoolto4°C

4. MeltonealiquotofPBS-agarose(microwave300Wforapprox.1min)andplaceitinthewater bath(makesuretoclosethelidtopreventdilutionduetocondensation)

5. Pre-coolenoughcuvettesenoughtohostallslidesandthehorizontalelectrophoresischamber to4°C

Cellexposure

The exposure conditions given below describe a control experiment for suspension cells and may be modiﬁed accordingto required experimental set-up. It should be noted that the exposure conditionsmayvarydependingonthe purposeofthestudy,e.g.,the typeofcellsused (celllineor

(5)

primary cells, adherentorsuspension), withorwithout metabolic activation, enzymatictreatments andexposuretimes.

1.Countthecellsanddeterminethecellviabilityusingtrypanblueexclusion;>95%isminimum levelatthispoint.

2.Transferto1.5mltesttubes(1× 10⁶ cells/tube)

3.Bringthetotalvolumeto990μlusingcellgrowthmedium

4.Add 10 μl exposure solution and incubate the tubes at 37°C with slight agitation for 3 h.

If hydrogen peroxide, ionizing radiation or other compound is used as positive exposure, incubationtimemaybeadjustedaccordingly.

Flash-comet

AllstepsfromthispointshouldbeperformedinanUV-lightprotectingenvironment(forexample, usingadarkroomandsecuritylight)asfastaspossiblewhilekeepingthesamplesrefrigerated(not above4°C).

1.Afterexposure,transferthecellsto4mlofpre-cooledwashsolution.

2.Centrifuge the cellsat230 G (rpc)for5min. Removethesupernatant andadd 5mlof cold washsolution. Note: Sincethe exposuremedium maycontainhighlevels ofagentsclassiﬁed as carcinogenic,mutagenic and/or toxicto reproduction(CMR agents), it should be collected anddisposedashazardouswaste.

3.Centrifugethecellsat230G(rpc)for5min.Add1mlofcoldwashsolution,countthecellsand determinethecell viability.Note:Weusually setthelowerlimit ofviabilityto80%usingthe trypanbluedye-exclusionassay.However,thislimitshouldbe regardedasarecommendation ratherthanarequirement.Theacceptablelevelofcytotoxicitymayvarydependingonthetype ofgenotoxicexposure(sometimesitisnessearytoallowhighercytotoxicity inordertodetect genotoxic agents)andalsodependingon whattype ofcytostaticparameter thatis used.Add 4mlofcoldwashsolutionandcentrifugethecellsat230G(rpc)for5min.

4.Removesupernatantandaddcalculatedvolume ofwashsolutionso thatcellconcentration is 0.8× 10⁶ (approx.100μl,butneverbelow50μl)

5.Whenallsamplesarediluted,placetheminawaterbathat37°Cforapprox.2–5min.

6.Aspirate thecell suspension 2–4times andtransfer 30μl ofthe cell suspension into a tube containing210μlagarose(donotmix,justtransfer).

7.Aspirate once and transfer 60 μl of agarose/cell suspension to a pre-heated randomized Gelbond^R coatedmicroscopeslide.Coverslidewithacoverslip(24× 40mm)

8.Placetheslideintherefrigeratororonacoldsurfacefor1–15mintoallowagarosetoset.(If usingGelBond^R,thisstepcanbereducedto1min)

9.When the agarosehasformed a gel,remove the coverslipandplace theslide inone ofthe pre-cooledcuvettes.Addcoldlysissolutionandincubatefor1hat4°C.

10.After celllysis,transfertheslideto thehorizontalelectrophoresischamberat4°C.Makesure toplacethegel-sideup.

11. Cover the slidewithcold electrophoresissolution andincubate for2.5min.After incubation, runelectrophoresisfor1minat150V(5V/cm)at4°C.

12. After electrophoresis transfer the cellsto a horizontalneutralization chamber and cover the slideswithcoldneutralizationsolutionandincubatefor15minat4°C.

13. After neutralization, dehydratethegelsby incubatinginEtOH(70%:95%:99%;2min/step)and letdry.

14.Storetheslidesinadark,dryanddust-freeenvironmentuntilanalysis.

Staining

Inordertopreventfadingandtoachievethebeststaining,itisrecommendedthatthestainingis conductedinanUVlightprotectiveenvironmentandawayfromdirectlightexposure.

1.Rehydratetheslidesin0.4MTris-HCL,pH7.5for15s

(6)

2. Add20μlofﬂuorescentstain(e.g.SYBR^R Gold;1:10,000in0.4M Tris-HCL)andincubatefor 10mininahumiditychamber

3. Washslides2× 1minin0.4MTris-HCL

4. Dab off excess liquid with paper and cover gel with cover slips (24 × 55 mm). Store in a humiditychambertopreventdrying.

Microscopy

Inordertopreventfadingandtoachievethebeststaining,itisrecommendedthatthemicroscopy isconductedinanUVlightprotectiveenvironmentandawayfromdirectlightexposure.

1. Analyzetheslidesinchronologicalorderaccordingtotherandomizationmethod(A-U;1–21or similar)

2. Analysis startsat approx. 3mm away from the slideedge andfollows the midline direction (seeFig.1)untilapprox.3mmfromthegeledge.

3. Fiftyrandomlychosennuclei/cometsarescoredoneach slide.Asidefromtheparametersthat areautomaticallyrecordedforeachmeasurement,itisalsoimportanttodocumentthenumber ofhedgehogs/clouds/ghosts,thebackgroundappearance(cleanorcluttered),celldensity(high or low), cell aggregation (high or low) and comet tail appearance (brush, granular, smooth, ﬁbrous).

4. If fewer than 50 comets are scored in one sweep, returnto starting position andmove one frame up/down, represented by the dashed arrows in Fig.1 (to avoidhaving the same cells scoredtwice).

Fig. 1. Analysis sweep over a comet slide.

Methodvalidation

InarecentlyacceptedpaperinMutationResearch[7],ournewFlash-cometandtheconventional cometassaywasusedwhenevaluatingtheDNAdamageinducedbyhydrogenperoxideandionizing radiation. As expected, both protocols the conventional comet assay and the Flash-comet assay showedastatisticallysigniﬁcantincreaseinH2O2-inducedDNAdamageatallconcentrationstested, evaluating the statistical signiﬁcance using either our recently published proportional odds model tailored to continuousoutcomes, which we havecalled UCDAS [8], ora more traditional, one-way ANOVA.However, whereastheFlash-cometshowedaclearconcentration dependentincreaseinthe H₂O₂-inducedDNA-damage,theincreaseinDNA-damagereachedaplateauat20μ^M^H2O₂ usingthe conventional protocol(Fig. 2A).The background level ofDNA-damage wasalso lower in theFlash- comet than in the traditional comet assay. Bothversions of the comet assay showed a clear dose response for radiation-induced radiation (Fig. 2B), but theslope ofthe dose-response curve in the Flash-cometshowedasteeperinclinecomparedtowhatwasseenintheconventionalassay.

Inthepresentpaper,wepresentaprotocolthatwehavecalledtheFlash-comet;anewalternative approachtotheconventionalcometassayunderalkalineconditions.Themostprofoundmodiﬁcation ofthe protocolforthe Flash-cometisthe introductionofa low-conductive electrophoresis solution based on LiOH at pH 12.5. This allows the Flash-cometassay to be conducted at six-times higher voltagecomparedtotheconventionalcometassay,leadingtoadramaticallyreducedelectrophoresis time and, as itseems, improved sensitivity. However, we donot claim that the times givenin the

(7)

Fig. 2. Panel A: TK-6 cells were exposed for 15 min to different concentration of either hydrogen peroxide (10–40 μM) or Milli-Q water as the negative control. After exposure, the cells were subjected to either the Flash-comet (white bars) or the conventional alkaline comet assay (dotted bars). The% TDNA was used as the indicator of DNA-damage. The data is presented as means ( ± 95% conﬁdence interval; CI) after pooling the data from three independent experiments ( n = 450 cells). Two different methods were used for the statistical analysis (i) a recently published proportional odds model tailored to continuous outcomes which we have called UCDAS ( 8 ) ( ^∗P < 0.05; ^∗∗∗P < 0.001) and (ii) a traditional one-way ANOVA with Tukey post- hoc test ( ††† P < 0.001). Panel B: In a proof of concept experiment, the% TDNA was measured using the Flash-comet (triangles) or the conventional alkaline comet assay (circles) on TK-6 cells irradiated with X-rays corresponding to 0, 2.5, 5 and 10 Gy. Both protocols showed a clear dose-response relationship in DNA-damage corresponding to an increased level of irradiation but the slope of dose-response curve for the Flash-comet showed a steeper incline. The data is presented as means ( ± 95% CI) after pooling the data from three slides ( n = 150 cells for each exposure).

variousstepsintheFlash-cometprotocolareabsolutelyoptimalandwedorecognizethattheFlash- comet assay alsohas to be further evaluated usingother positive controlsthan H₂O₂ andionizing radiation.

DeclarationofCompetingInterest

The authors declare that they have no known competing ﬁnancial interests or personal relationshipsthatcouldhaveappearedtoinﬂuencetheworkreportedinthispaper.

Acknowledgments

The authors are indebted to Ms. Lena Norgren forher wonderful service andtechnical support through manyyears.WewouldalsoliketogratefullyacknowledgeDr.HåkanBivehedforhisvalued andinsightfulinputonthechemicalaspectsofthepresentedprotocol,andProfessorBoStenerlöwfor allowingustousehisfacilitiesfortheradiationexperiments.Finally,wewouldliketothankProfessor Ola Söderberg, aco-author inourpaperpublishedin MutationResearch [7],helpingusto perform some of theproof-of-concept experiments regarding the stabilityof DNA atdifferent temperatures andpHconditions.

References

[1] O. Ostling , K.J. Johanson , Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells, Biochem. Biophys. Res. Commun. 123 (1984) 291–298 .

[2] N.P. Singh , M.T. McCoy , R.R. Tice , E.L. Schneider , A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell Res. 175 (1988) 184–191 .

[3] A . Azqueta, A .R. Collins, The essential comet assay: a comprehensive guide to measuring DNA damage and repair, Arch.

Toxicol. 87 (2013) 94 9–96 8. https://doi.org/10.10 07/s0 0204- 013- 1070- 0 .

[4] A .R. Collins, A .A . Oscoz, G. Brunborg, I. Gaivao, L. Giovannelli, M. Kruszewski, C.C. Smith, R. Stetina, The comet assay: topical issues, Mutagenesis 23 (2008) 143–151. https://doi.org/10.1093/mutage/gem051 .

[5] S.A. Shaposhnikov, V.B. Salenko, G. Brunborg, J. Nygren, A.R. Collins, Single-cell gel electrophoresis (the comet assay): loops or fragments, Electrophoresis 29 (2008) 3005–3012. https://doi.org/10.1002/elps.200700921 .

[6] P.L. Olive, J.P. Banath, The comet assay: a method to measure DNA damage in individual cells, Nat. Protoc. 1 (2006) 23–29.

https://doi.org/10.1038/nprot.2006.5 .

(8)

[7] E. Bivehed, O. Söderberg, B. Hellman, Flash-comet: signiﬁcantly improved speed and sensitivity of the comet assay through the introduction of lithium-based solutions and a more gentle lysis, Mutat. Res. Genet. Toxicol. Environ. Mutagen (2020) 858–860 503240. https://doi.org/10.1016/j.mrgentox.2020.503240 .

[8] E. Bivehed, A. Gustafsson, A. Berglund, B. Hellman, Evaluation of potential DNA-damaging effects of nitenpyram and imidacloprid in human U937-cells using a new statistical approach to analyse comet data, Expos. Health 12 (2020) 547–554.

https://doi.org/10.1007/s12403- 019- 00328- 6 .