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AntibodiesOnline/CytoSelect™ 96Well Cell Transformation Assay (Cell Recovery Compatible, Fluoromet188bio精品生物—专注于实验室精品爆款的电商平台 - 蚂蚁淘旗下精选188款生物医学科研用品
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AntibodiesOnline/CytoSelect™ 96Well Cell Transformation Assay (Cell Recovery Compatible, Fluoromet
Reactivity
Mammalian
Application
CellularAssay(CA)
Options
Bulkdiscount
Supplier
SupplierProductNo.
BrandCytoSelect™
SampleTypeCellSamples
DetectionMethodFluorometric
CharacteristicsCytoSelect™CellTransformationAssaydoesnotinvolvesubjectivemanualcountingofcoloniesorrequirea3-4weekincubationperiod.Insteadcellsareincubatedonly6-8daysinaproprietarysemisolidagarmediabeforebeingsolubilized,lysedanddetectedbythepatentedCyQuant®GRDyeinafluorescenceplatereader(seeAssayPrinciplebelow).Alternatively,viabletransformedcellscanbeeasilyrecoveredforfurtherculturingandtesting,suchasinprotein/DNAarrayanalysisandcancervaccinedevelopment.Thisformatprovidesaquantitative,high-throughputmethodtoaccuratelymeasurecelltransformation,whiletheshortincubationtimemakesitpossibletoassaycellstransientlytransfectedwithoncogenesorsiRNA.TheCytoSelect™CellTransformationKit(CellRecoveryCompatible)providesarobustsystemformeasuringinvitrodrugsensitivity,screeningoncogenesandcelltransformationinhibitors,andallowsfortransformedcellrecovery.EachTrialSizekitprovidessufficientquantitiestoperform24assaysina96-wellplate,12assaysina48-wellplate,6assaysina24-wellplate,or3assaysina12-wellplate.2
Components
  1. 10XCytoSelect™AgarMatrixSolution:One1mLsteriletube
  2. CytoSelect™MatrixDiluent:One1mLsteriletube
  3. 5XDMEMSolution:One1.5mLsteriletube
  4. 10XMatrixSolubilizationSolution:One0.6mLsteriletube
  5. 4XLysisBuffer:One2mLtube
  6. CyQuant®GRDye:One25μLtube
Materialnotincluded
  1. CellsandCultureMedium
  2. 37°CIncubator,5%CO2Atmosphere
  3. LightMicroscope
  4. 96-wellFluorometer
  5. 37°Candboilingwaterbaths
  6. (Optional)PositiveControlcellssuchasNIH3T3(RasG12V)
BackgroundNeoplastictransformationoccursviaaseriesofgeneticandepigeneticalterationsthatyieldacellpopulationthatiscapableofproliferatingindependentlyofbothexternalandinternalsignalsthatnormallyrestraingrowth.Forexample,transformedcellsshowreducedrequirementsforextracellulargrowthpromotingfactors,arenotrestrictedbycell-cellcontact,andareoftenimmortal.Anchorage-independentgrowthisoneofthehallmarksoftransformation,whichisconsideredthemostaccurateandstringentinvitroassayfordetectingmalignanttransformationofcells.Traditionally,thesoftagarcolonyformationassayisacommonmethodtomonitoranchorage-independentgrowth,whichmeasuresproliferationinasemisolidculturemediaafter3-4weeksbymanualcountingofcolonies.Standardsoftagarassaysareusuallyperformedin100-mmor60mmdishes,wherecellsareallowedtogrowinsideasemisolidculturemediafor3-4weeksbeforesizablecoloniesappear.Thismethodisquitecumbersome,time-consuming,anddifficultwhentestingalargenumberofsamples.Additionally,themanualcountingofcoloniesishighlysubjective,withvaryingcolonysizes,itsdifficulttodeterminemeaningfulresults.
ApplicationNotesOptimalworkingdilutionshouldbedeterminedbytheinvestigator.
Comment

  • Proprietarymodifiedsoftagarmedium
  • Fullyquantifycelltransformationwithnomanualcellcounting
  • Resultsin7-8days,not3weeks
  • Recovercellsfromsoftagarmediumforfurtherdownstreamanalysis

AssayTime7-8d
ReagentPreparation
  • 2XDMEM/20 %FBSMedium:Inasteriletube,dilutetheprovided5XDMEMinsterilecellculturegradewaterto2Xcontaining20 %FBS.Forexample,topreparea2.5 mLsolution,add1 mLof5XDMEM,0.5 mLofFBSand1 mLofsterilecellculturegradewater.Sterilefilterthe2Xmediato0.2μm.Note:YoumaysubstituteyourownmediuminplaceoftheDMEMweprovide,butensurethatitisata2Xconcentration.
  • 1XMatrixSolubilizationSolution:Preparea1XMatrixSolubilizationSolutionbydilutingtheprovided10Xstock1:10insterilecellculturegradewater.Sterilefilterthe1Xsolutionto0.2μm.
  • 10XCytoSelect™AgarMatrixSolution:HeattheAgarMatrixSolutiontubeto90-95 °Cinawaterbathfor30 minutes,oruntilagarmatrixliquefies.Transferthetubetoa37 °Cwaterbathfor20 minutesandmaintainuntilneeded.4
AssayProcedure

Thefollowingassayprotocoliswrittenfora96-wellformat.Refertothebelowtablefortheappropriatedispensingvolumesofotherplateformats.CultureDish96-well48-well24-well12-well6-wellBaseAgarMatrixLayer501002505001000(μL/well)CellSuspension/Agar751503757501500MatrixLayer(μL/well)CultureMedia(μL/well)5010025050010001XMatrixSolubilizationSolution12525062512502500(μL/well)Table

  1. DispensingVolumesofDifferentPlateFormats

    I.PreparationofBaseAgarMatrixLayer

    1. Heatthe10XCytoSelect™AgarMatrixSolutionto90-95 °Cinawaterbathfor30 minutes,oruntilagarmatrixliquefies.Transferthetubetoa37 °Cwaterbathfor20 minutesandmaintainuntilneeded.
    2. Warmthe2XDMEM/20 %FBSmedium(seePreparationofReagentssection)to37 °Cinawaterbath.Allowatleast30 minutesforthetemperaturetoequilibrate.
    3. AccordingtoTable2(below),preparethedesiredvolumeofBaseAgarMatrixLayerinthefollowingsequence:a.Inasteriletube,addtheappropriatevolumeof2XDMEM/20 %FBSmedium.b.Next,addthecorrespondingvolumeofsterilewater.Mixwell.c.Finally,addthecorrespondingvolumeof10XCytoSelect™AgarMatrixSolution.Mixwell.Note:The10XCytoSelect™AgarMatrixSolutionisslightlyviscous,careshouldbetakeninaccuratelypipettingtheappropriatevolume.2XDMEM/20 %SterileWater10XTotalVolumeof#ofTestsin96-FBSMedium(mL)CytoSelect™BaseAgarMatrixwellPlate(50(mL)AgarMatrixLayer(mL)μL/test)Solution(mL)0.6250.50.1251.2525Table
    4. PreparationofBaseAgarMatrixLayer
    5. Aftermixing,maintaintheBaseAgarMatrixLayerat37 °Ctoavoidgelation.5
    6. Dispense50μLofBaseAgarMatrixLayerintoeachwellofa96-wellsterileflat-bottommicroplate(samplesshouldbeassayedintriplicate).GentlytaptheplateafewtimestoensuretheBaseAgarMatrixLayerevenlycoversthewells.Notes:•Workquicklywiththelayertoavoidgelation.Also,trytoavoidaddingairbubblestothewell.•Toavoidfastandunevenevaporationthatleadstoaberrantresults,wesuggestnotusingthewellsontheplateedge,orfillingtheedgewellswithmediumtoreduceevaporation.
    7. Transfertheplateto4 °Cfor30 minutestoallowtheBaseAgarMatrixLayertosolidify.
    8. PriortoaddingtheCellSuspension/AgarMatrixLayer(SectionII),allowtheplatetowarmtoroomtemperaturefor30 minutes.

    II.AdditionofCellSuspension/AgarMatrixLayer(understerileconditions)

    1. Heatthe10XCytoSelect™AgarMatrixSolutionto90-95 °Cinawaterbathfor30 minutes,oruntilagarmatrixliquefies.Transferthetubetoa37 °Cwaterbathfor20 minutesandmaintainuntilneeded.
    2. Warmthe2XDMEM/20 %FBSmedium(seePreparationofReagentssection)andCytoSelect™MatrixDiluentto37 °Cinawaterbath.Allowatleast30 minutesforthetemperaturetoequilibrate.
    3. Harvestandresuspendcellsinculturemediumat0.1-1x106cells/mL.Keepthecellsuspensionwarmina37 °Cwaterbath.
    4. AccordingtoTable3(below),preparethedesiredvolumeofCellSuspension/AgarMatrixLayerinthefollowingsequence:a.Inasteriletube,addtheappropriatevolumeof2XDMEM/20 %FBSmedium.b.Next,addthecorrespondingvolumeofCytoSelect™MatrixDiluent.Mixwell.c.Next,addthecorrespondingvolumeof10XCytoSelect™AgarMatrixSolution.Mixwell.d.Finally,addthecorrespondingvolumeofcellsuspension.Mixwell.Note:TheCytoSelect™MatrixDiluentand10XCytoSelect™AgarMatrixSolutionareslightlyviscous,careshouldbetakeninaccuratelypipettingtheappropriatevolumes.2XCytoSelect™10XCellTotalVolumeof#ofTestsinDMEM/20 %MatrixDiluentCytoSelect™SuspensionCellSuspension/96-wellPlateFBSMedium(mL)AgarMatrix(mL)AgarMatrix(75μL/test)(mL)Solution(mL)Layer(mL)0.8750.6880.1880.1251.87525Table
    5. PreparationofCellSuspension/AgarMatrixLayer6
    6. Aftermixing,incubatetheCellSuspension/AgarMatrixLayeratroomtemperaturefor5 minutes.
    7. Immediatelydispense75μLofCellSuspension/AgarMatrixLayerintoeachwellofthe96-wellplate,alreadycontainingtheBaseAgarMatrixLayer(SectionI).Notes:•Workquicklywiththelayertoavoidgelation,butgentlypipetteasnottodisruptthebaselayerintegrity.Also,trytoavoidaddingairbubblestothewell.•AlwaysincludenegativecontrolwellsthatcontainnocellsintheCellSuspension/AgarMatrixLayer.
    8. Transfertheplateto4 °Cfor20 minutestoallowtheCellSuspension/AgarMatrixLayertosolidify.
    9. Allowtheplatetowarmtoroomtemperaturefor30 minutes.
    10. Add50μLofculturemediumcontainingcellgrowthactivator(s)orinhibitor(s)toeachwell.
    11. Incubatethecellsfor6-8daysat37 °Cand5 %CO2.Examinethecolonyformationunderalightmicroscope.

    III.QuantitationofAnchorage-IndependentGrowth(skiptosectionIVifcellrecovery/re-platingisdesired)

    1. Add125μLof1XMatrixSolubilizationSolutiontoeachwell.
    2. Pipettetheentirevolumeofthewell10-12timestomixthoroughlyandsolubilizetheagarmatrixcompletely.
    3. Transfer150μLofthemixturetoa96-wellplatesuitableforfluorescencemeasurement.
    4. Preparesufficient4XLysisBuffer/CyQuant®GRdyesolutionforallsamplesbydilutingthedye1:75in4XLysisBuffer(forexample,add5μLdyeto370μLof4XLysisBuffer).
    5. Add50μLof4XLysisBuffer/CyQuant®GRdyesolutiontoeachwell(alreadycontaining150μLofsolution).Incubatetheplateatroomtemperaturefor30 minutes.
    6. Pipetteeachwell7-10timestoensureahomogeneousmixture.
    7. Readtheplateina96-wellfluorometerusinga485/520nmfilterset.

    IV.CellRecoveryandRe-plating(understerileconditions)

    1. Add125μLof1XMatrixSolubilizationSolutiontoeachwell.
    2. Pipetteeachwell10-12timestomixthoroughly.
    3. Transfertheentiremixturetoatleast20volumesofstandardculturemedium(forexample,1 mLwouldbetransferredto20 mLmedia).
    4. Pipettethemixturevigorously7-10times.
    5. Centrifugethecellpelletandaspiratethemediasupernatant.7
    6. Resuspendthecellpelletinanother20volumesofstandardculturemedium.
    7. Repeatsteps4-6.
    8. Resuspendthepelletandtransfertoatissuecultureflaskordish.
    9. Transfertoacellcultureincubator.CellDoseCurve(optional)
      1. Heatthe10XCytoSelect™AgarMatrixSolutionto90-95 °Cinawaterbathfor30 minutes,oruntilagarmatrixliquefies.Transferthetubetoa37 °Cwaterbathfor20 minutesandmaintainuntilneeded.
      2. Warmthe2XDMEM/20 %FBSmedium(seePreparationofReagentssection)andCytoSelect™MatrixDiluentto37 °Cinawaterbath.Allowatleast30 minutesforthetemperaturetoequilibrate.
      3. Harvestandresuspendcellsinculturemediumat1-5x106cells/mL.
      4. Prepareaserial2-folddilutioninculturemedium,includingablankwithoutcells.
      5. Transfer50μLofeachdilutiontoa96-wellplate.
      6. AccordingtoTable4(below),preparethedesiredvolumeofCellDoseCurveSolutioninthefollowingsequence:a.Inasteriletube,addtheappropriatevolumeof2XDMEM/20 %FBSmedium.b.Next,addthecorrespondingvolumeofsterilewater.Mixwell.c.Next,addthecorrespondingvolumeofCytoSelect™MatrixDiluent.Mixwell.d.Finally,addthecorrespondingvolumeof10XCytoSelect™AgarMatrixSolution.Mixwell.Note:TheCytoSelect™MatrixDiluentand10XCytoSelect™AgarMatrixSolutionareslightlyviscous,careshouldbetakeninaccuratelypipettingtheappropriatevolumes.2XDMEM/20 %SterileWaterCytoSelect™10XCytoSelect™TotalVolumeofFBSMedium(mL)MatrixDiluentAgarMatrixCellDoseCurve(mL)(mL)Solution(mL)Solution(mL)1.250.450.550.252.50.6250.2250.2750.1251.25Table
      7. PreparationofCellDoseCurveSolution
      8. Immediatelydispense125μLofCellDoseCurveSolutionintothewellsofthe96-wellplate,alreadycontainingthecellserialdilution(fromstep5).
      9. Add125μLof1XMatrixSolubilizationSolutiontoeachwell.Pipetteeachwell10-12timestomixthoroughly.
      10. Transfer150μLofthemixturetoa96-wellplatesuitableforfluorescencemeasurement.
      11. Preparesufficient4XLysisBuffer/CyQuant®GRdyesolutionforallsamplesbydilutingthedye1:75in4XLysisBuffer(forexample,add5μLdyeto370μLof4XLysisBuffer).8
      12. Add50μLof4XLysisBuffer/CyQuant®GRdyesolutiontoeachwell(alreadycontaining150μLofsolution).Incubatetheplateatroomtemperaturefor30 minutes.
      13. Pipetteeachwell7-10timestoensureahomogeneousmixture.
      14. Readtheplateina96-wellfluorometerusinga485/520nmfilterset.

CalculationofResults
  1. CompareRFUvalueswiththeCellDoseCurveandextrapolatethecellconcentration.
  2. CalculatetheTotalTransformedCellNumber/WellTotalTransformedCells/Well=cells/mLx0.050 mL/wellForexample:IfyouextrapolateyourRFUvaluefromyourcelldosecurveanddetermineyouhave500,000cells/mLinyoursample.TotalTransformedCells/Well=500,000cells/mLx0.050 mL/well=25,000cells/well
RestrictionsForResearchUseonly
Storage4°C
StorageCommentStoreallcomponentsat4°C.
SupplierImages
Cellular Assay (CA) image for CytoSelect™ 96-Well Cell Transformation Assay (Cell Recovery Compatible, Fluorometric) (ABIN2344889)ViabilityofRecoveredCells. HeLaand293cellswereculturedfor6daysaccordingt...
Productcitedin:Montalbano,Curcurù,Shirafkan,Vento,Rastellini,Cicalese:"ModelingofHepatocytesProliferationIsolatedfromProximalandDistalZonesfromHumanHepatocellularCarcinomaLesion."in:PLoSONE,Vol.11,Issue4,pp.e0153613,2016(PubMed).


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