Alstembio/HumaniPSCLine,Episomal(MSC)/iPS12,Alstembio,,Alstembio,ALSTEM,+,,Array.Array.Array蚂蚁淘厂家直采.

产品名称：Alstembio/HumaniPSCLine,Episomal(MSC)/iPS12

产品编号：iPS12

市场价：¥25800.00

场地：美国(厂家直采)

产品分类：细胞相关>细胞研究>细胞代谢>

联系QQ：1570468124

电话号码：4000-520-616

邮箱： info@ebiomall.com

美元价：$1290.00

品牌： Alstembio

公司：ALSTEM Inc

公司分类：

商品介绍

HumaniPSCellLine(Episomal,MSC):#iPS12

ProductDescription

Footprint-freehumaniPS(inducedpluripotentstem)celllinewasderivedfromhumanmesenchymalstromalcells(MSCs)byectopicexpressionofOCT4,SOX2,KLF4,andL-MYCgenesusingepisomalplasmids.ThecellswerederivedusingmorphologicalselectioncriteriaandwithouttheuseoffluorescentMarkerordrugselection.WhenculturedunderstandardhumanEScellcultureconditions,themorphologyofhumaniPScellsareidenticaltothatofhumanEScells.ThecellsalsoexpressthepluripotencymarkersTRA-1-60,SSEA-3andOct4,anddemonstratestrongendogenousalkalinephosphataseactivity.

ImmunoStaining

Figure1.CharacterizationofiPSCsderivedfromhumanmesenchymalstromalcells(MSCs)usingALSTEMepisomalvectors.AlkalinephosphatasestainingofiPSCclone,immunostainingofcoloniesexpressingESCspecificmarkersOCT4andTRA-1-60.

ProductSpecifications
ProductName	HumaniPSCLine,Episomal(MSC)
Catalog#	iPS12
Size	2x10⁵cells/vial
Shipping	DryIce
StorageandStABIlity	Storeingasphaseofliquidnitrogenimmediatelyuponreceipt.Thisproductisstablefor6monthswhenstoredasdirected.
QualityControl	HumaniPScellsweregrowninfeederfreeconditionswithmTeSR1medium.EachlotofhumaniPScellsistestedforgrowthandviabilityfollowingrecoveryfromcryopreservation.Inaddition,eachlotistestedforexpressionofTRA-1-60andOct4,aswellastheactivityofalkalinephosphatase.
SafetyPrecaution	ALSTEMhighlyrecommendsthatprotectivegloves,alabcoat,andafull-facemaskalwaysbewornwhenhandlingfrozenvials.Itisimportanttonotethatsomeliquidnitrogencanleakintothevialswhensubmersedinliquidnitrogen.Uponthawing,theliquidnitrogenreturnstothegasphase,resultinginexcessivepressurewithinthevialthatcancausethevialtoexplodeorexpelthecapwithdangerousforce.
RestrictedUse	ForResearchUseOnly.Notforuseindiagnosticortherapeuticprocedures.
Protocol	Download

Overview
Procedure

ThisprotocolcanbeusedforculturinghumaniPScells.Footprint-freehumaniPScellsweregeneratedbytransientlyintroducingepisomalplasmidsencodingthehumantranscriptionfactorsintohumanmesenchymalstromalcells(MSCs).Thecellswerederivedusingmorphologicalselectioncriteriaandwithouttheuseoffluorescentmarkersordrugselection.WhenculturedunderstandardhumanEScellcultureconditions,themorphologyoffootprint-freehumaniPScellsisidenticaltothatofhumanEScells.ThecellsalsoexpressthepluripotencymarkersTRA-1-60andOct4,anddemonstrateastrongendogenousAPactivity.

I.Feederfreecultureconditions

Preparationoffeeder-freemedium

ThawmTeSR15XSupplement(Cat.no.05850,STEMCELLTechnologies)atroomtemperatureorovernightat4°C.
Addthe100mLofthawed5XSupplementto400mLBasalMediumforatotalvolumeof500mLaseptically.Mixwellandfilterthrougha0.2μm,low-proteinbindingfilter,ifdesired.
Aliquotintoappropriateamountaccordingtousageandstorethealiquotsat4°C.

CoatingplateswithMatrigel

Matrigel(Cat.no.354277,BD)shouldbealiquotedandstoredat-80°Cforlong-termuse.

Thawmatrigeloniceuntilliquid.Dilutematrigel1:30to1:50withpre-chilledKODMEM/F12.
Immediatelyusethedilutedmatrigelsolutiontocoattissueculture-treatedplates.Fora6-wellplate,use1mLofdilutedmatrigelsolutionperwell,andswirltheplatetospreadthematrigelsolutionevenlyacrossthesurface.
Letthecoatedplatestandfor1hat37°Corovernightat4°C.Ifplatehasbeenstoredat4°C,allowtheplatetoincubateat37°Cforatleast30minutesbeforeremovingthematrigelsolution.

ThawingcryopreservedhumaniPScells

QuicklythawthehumaniPScellsina37°Cwaterbathbygentlyshakingthecryovialcontinuouslyuntilhalfthawed.Removethecryovialfromthewaterbathandspraywith70%ethanoltosterilize.
Transferthecontentsofthecryovialtoa15mLconicaltube.Add5mLwarmmTeSR1dropwisetothetube,gentlymixingasthemediumisadded.
Centrifugecellsat200xgfor5minutesatroomtemperature.
Aftercentrifugation,aspiratethemediumfrom15mLtube.GentlyresUSPendthecellpelletin2mLmTeSR1with5µMROCKinhibitor,takingcaretomaintainthecellsassmallcellclumps.
Removethematrigelsolutionfromacoatedtissueculture6-wellplate.Transferthemediumcontainingthecellclumpstothematrigelcoated6-wellplate.
Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO₂and95%humidity.
Changemediumdaily.Checkforundifferentiatedcoloniesthatarereadytopassagewhencoloniesarebigenough(approximately7-10daysafterthawing).

PassaginghumaniPScellsgrownunderfeeder-freeconditions

Useamicroscopetoidentifyregionsofdifferentiation.Markthedifferentiatedcoloniesusinglensmarkeronthebottomoftheplate.
RemoveregionsofdifferentiationbyscrapingwithaPipettetiporbyaspiration.
AspiratemediumfromthehumaniPScellcultureandrinsewithDPBS(2mL/well).
Add1mLperwellofEZStemEnzyme-FreeStemCellDissociationSolution(cat.no.M100,ALSTEM),andincubateat37°Cfor2-3minutes.Oradd0.5mLperwellofaccutase(Cat.no.SCR005,Millipore,diluted1:1withDPBSbeforeuse),andincubateat37°Cfor1minute.
RemoveEZStemEnzyme-FreeStemCellDissociationSolutionoraccutase,gentlyrinseeachwell2-3timeswith2mLofDMEM/F-12perwellandtransferthedetachedcellaggregatestoa15mLconicaltube.
Add2mL/wellmTeSR1andscrapecoloniesoffwithacelllifter.Transferthedetachedcellaggregatestoa15mLconicaltube.
Rinsethewellwithanadditional2mLmTeSR1tocollectanyremainingaggregates.Addtherinsetothe15mLtube.
Centrifugethe15mLtubecontainingtheaggregatesat200xgfor5minutesatroomtemperature.
Aspiratethesupernatant.ResuspendpelletinmTeSR1containing5µMROCKinhibitorbygentlypipettingandensurethatcellsaremaintainedasaggregates.
PlatethehumaniPScellaggregateswithmTeSR1inanewplatecoatedwithmatrigel.(Removematrigelsolutionbeforeplating).
Note:Ifthecoloniesareatanoptimaldensity,thecellscanbesplitevery5-7daysusing1:3to1:6ratios.
Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO₂and95%humidity.
Changemediumdaily.

CryopreservinghumaniPScells

PrepareEZStemfreezingmedium(Cat.no.M050,ALSTEM)onice.
Performsteps1-8fromPassaginghumaniPScellsgrownunderfeeder-freeconditions.
Gentlyaspiratethesupernatantandloosenthecellpelletbytappingthebottomofthetube.
Gentlyresuspendthepelletinfreezingmedium,takingcaretoleavetheclumpslargerthanthatwouldnormallybedoneforpassaging.
Transfer1mLofclumpsinfreezingmediumintoeachlabeledcryogenicvial.
Placevialsintoafreezingcontainerandplacethecontainerat-80°Covernight.
Transfertoaliquidnitrogentanknextday.

II.Feeder-dependentcultureconditions

PreparationofhumanESmedium

KnockoutDMEM/F12containing20%knockoutserumreplacement,2mMglutamine,0.1mMnonessentialaminoacids,0.1mM2-mercaptoethanol,10ng/mlbFGF,and50Uand50µg/mlpenicillinandstreptomycin.

ThawingcryopreservedhumaniPScells

Toinsurethehighestlevelofviability,besuretowarmmediumto37°Cbeforeusingonthecells.DuetothelowsurvivalrateofcryopreservedhumaniPScells,therecoveryisexpectedtotakeatleastoneweek.

QuicklythawthehumaniPScellsina37°Cwaterbathbygentlyshakingthecryovialcontinuouslyuntilhalfthawed.Removethecryovialfromthewaterbathandspraywith70%ethanoltosterilize.
Transferthecontentsofthecryovialtoa15mLconicaltube.Add5mLwarmhumanESmediumdropwisetothetube,gentlymixingasthemediumisadded.
Centrifugecellsat200xgfor5minutesatroomtemperature.
Whilecentrifuging,removeMEFmediumfromthefeedercellplates,andwashthewellstwicewithKnockoutDMEM/F12.Thenadd1mlofhumanESMediumwith5µMROCKinhibitor(Y-27632,StemRD)toonewellof6-wellplate.
Aftercentrifugation,aspiratethemediumfrom15mLtube.Gentlyresuspendthecellpelletin1mLfreshhumanESmediumcontaining5µMROCKinhibitor(Y-27632),takingcaretomaintainthecellsassmallcellclumps.
Transferthemediumcontainingthecellclumpstoonewellof6-wellplatewithMEFfeedercells.
Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO₂and95%humidity.
Changemediumdaily.Checkforundifferentiatedcoloniesthatarereadytopassagewhencoloniesarebigenough(approximately7-10daysafterthawing).

PassaginghumaniPScellsgrownunderfeeder-dependentconditions

Aspiratethemediumandwashthecellstwicewith1mlofPBS.
RemovePBScompletely,add0.5mlofAccutase(Cat.no.SCR005,Millipore,diluted1:1withDPBSbeforeuse)andincubatefor1-2minat37°C.
Tapthebottomoftheplatetodislodgethecellsfromthebottomoftheplate.Thenaspiratetheaccutase.
Add1mlofDMEM/F12totheplateandcarefullywashthefeedercells,andaspiratethemedium.Repeat.
Add1mlofhumanESmediumcontaining5µMROCKinhibitortotheplateandsuspendthecellcoloniesbygentlypipettingupanddown.Itisimportantnottobreakupthecoloniesintosinglecells.
RemoveaplateofMEFfeedercellsfromtheincubator.AspiratetheMEFmedium.WashoncewithKODMEM/F12medium.
Distribute0.2-0.3mlofthehumaniPScellsuspensiontoeachwellofa6-wellplate.
Add1mLhumanESmediumtotheoriginalwellandscrapecoloniesoffwithacelllifter.
Distribute0.2-0.3mlofthehumaniPScellsuspensiontoeachwellofa6-wellplate.
AddhumanESmediumwithROCKinhibitortoafinalvolumeof2mlperwell.RightafterplatingtheiPScells,gentlyswirltheplateback-and-forthandside-to-sideandincubateat37°C.

Note:Ifthecoloniesareatanoptimaldensity,thecellscanbesplitevery5-7daysusing1:3to1:6ratios.

After24hours,removethemediaandreplacewithhumanESmedia(withoutROCKinhibitor).
ThehumanESmediamustbechangedeverydayandhumaniPScellssubculturedevery5-7days.Trackthepassagenumberofthecells.

CryopreservinghumaniPScells

PrepareEZStemfreezingmedium(Cat.no.M050,ALSTEM)onice.
Performsteps1-6fromPassaginghumaniPScellsgrownunderfeeder-dependentconditions.
Transferthedetachedcellaggregatestoa15mLconicaltube.
Add1mLhumanESmediumandscrapecoloniesoffwithacelllifter.
Transferthecellsuspensiontothe15mLconicaltube.
Centrifugethe15mLtubecontainingtheaggregatesat200xgfor5minutesatroomtemperature.
Gentlyaspiratethesupernatantandloosenthecellpelletbytappingthebottomofthetube.
Gentlyresuspendthepelletinfreezingmedium,takingcaretoleavetheclumpslargerthanthatwouldnormallybedoneforpassaging.
Transfer1mLofclumpsinfreezingmediumintoeachlabeledcryogenicvial.
Placevialsintoanisopropanolfreezingcontainerandplacethecontainerat-80°Covernight.
Transfertoaliquidnitrogentanknextday.

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