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蚂蚁淘在线 / 品牌中心 / Alstembio / Alstembio/HumaniPSCLine,Episomal/iPS11

Alstembio/HumaniPSCLine,Episomal/iPS11

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￥25800.00

货号：iPS11

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品牌：Alstembio

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商品描述

HumaniPSCellLine(Episomal,HFF):#iPS11

ProductDescription

Footprint-freehumaniPS(inducedpluripotentstem)celllinederivedfromhumanforeskinfibroblasts(HFFs)byectopicexpressionofOCT4,SOX2,KLF4,andc-MYCgenesusingepisomalplasmids.ThecellswerederivedusingmorphologicalselectioncriteriaandwithouttheuseoffluorescentMarkerordrugselection.WhenculturedunderstandardhumanEScellcultureconditions,themorphologyofhumaniPScellsareidenticaltothatofhumanEScells.ThecellsalsoexpressthepluripotencymarkersSSEA-3andNanog,anddemonstratestrongendogenousalkalinephosphatase.HighviABIlity,lowpassageiPScellshavebeenpre-adaptedtoserum-free,feeder-freecultureconditions.

ImmunoStaining

Figure1.CharacterizationofiPSCsderivedfromhumandermalfibroblastsusingALSTEMepisomalvectors.BrightfieldimageofhiPSCcolonies(topleft),immunostainingofhiPSCcoloniesexpressingESCspecificmarkersOCT4,SSEA-3,andTRA-1-81.

ProductSpecifications
ProductName	HumaniPSCLine,Episomal
Catalog#	iPS11
Size	2x10⁵cells/vial
Shipping	DryIce
StorageandStability	Storeingasphaseofliquidnitrogenimmediatelyuponreceipt.Thisproductisstablefor6monthswhenstoredasdirected.
QualityControl	HumaniPScells(cat.no.iPS11)weregrowninmTeSR1medium.EachlotofhumaniPScellsistestedforgrowthandviabilityfollowingrecoveryfromcryopreservation.Inaddition,eachlotistestedforexpressionofSSEA-3andNanog,aswellastheactivityofalkalinephosphatase.
SafetyPrecaution	ALSTEMhighlyrecommendsthatprotectivegloves,alabcoat,andafull-facemaskalwaysbewornwhenhandlingfrozenvials.Itisimportanttonotethatsomeliquidnitrogencanleakintothevialswhensubmersedinliquidnitrogen.Uponthawing,theliquidnitrogenreturnstothegasphase,resultinginexcessivepressurewithinthevialthatcancausethevialtoexplodeorexpelthecapwithdangerousforce.
RestrictedUse	ForResearchUseOnly.Notforuseindiagnosticortherapeuticprocedures.
Protocol	Download

Overview
Procedure

ThisprotocolcanbeusedforculturinghumaniPScells.Footprint-freehumaniPScellsweregeneratedbytransientlyintroducingepisomalplasmidsencodingthehumantranscriptionfactorsintohumanforeskinfibroblasts.Thecellswerederivedusingmorphologicalselectioncriteriaandwithouttheuseoffluorescentmarkersordrugselection.WhenculturedunderstandardhumanEScellcultureconditions,themorphologyoffootprint-freehumaniPScellsisidenticaltothatofhumanEScells.ThecellsalsoexpressthepluripotencymarkersSSEA-4andNanog,anddemonstrateastrongendogenousAPactivity.

Ⅰ.Feederfreecultureconditions

Preparationoffeeder-freemedium

1.ThawmTeSR15XSupplement(Cat.no.05850,STEMCELLTechnologies)atroomtemperatureorovernightat4°C.

2.Addthe100mLofthawed5XSupplementto400mLBasalMediumforatotalvolumeof500mLaseptically.Mixwell.Filterthrougha0.2μm,low-proteinbindingfilter,ifdesired.

3.Aliquotintoappropriateamountaccordingtousageandstorethealiquotsat4°C.

CoatingplateswithMatrigel

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

1.Thawmatrigeloniceuntilliquid.Dilutematrigel1:50withpre-chilledKODMEM/F12.

2.Immediatelyusethedilutedmatrigelsolutiontocoattissueculture-treatedplates.Fora6-wellplate,use0.8mLofdilutedmatrigelsolutionperwell,andswirltheplatetospreadthematrigelsolutionevenlyacrossthesurface.

3.Letthecoatedplatestandfor1hat37°Corovernightat4°C.Ifplatehasbeenstoredat4°C,allowtheplatetoincubateat37°Cforatleast30minutesbeforeremovingthematrigelsolution.

ThawingcryopreservedhumaniPScells

1.QuicklythawthehumaniPScellsina37°Cwaterbathbygentlyshakingthecryovialcontinuouslyuntilhalfthawed.Removethecryovialfromthewaterbathandspraywith70%ethanoltosterilize.

2.Transferthecontentsofthecryovialtoa15mLconicaltube.Add5mLwarmmTeSR1dropwisetothetube,gentlymixingasthemediumisadded.

3.Centrifugecellsat200xgfor5minutesatroomtemperature.

4.Whilecentrifuging,removethematrigelsolutionfromacoatedtissueculture6-wellplate.Add1mLofwarmmTeSR1containing10µMROCKinhibitor(Y-27632)toonewellof6-wellplate.

5.Aftercentrifugation,aspiratethemediumfrom15mLtube.GentlyresUSPendthecellpelletin1mLmTeSR1with10µMROCKinhibitor,takingcaretomaintainthecellsassmallcellclumps.

6.Transferthemediumcontainingthecellclumpstothematrigelcoated6-wellplate.

7.Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO2and95%humidity.

8.Changemediumdaily.Checkforundifferentiatedcoloniesthatarereadytopassagewhencoloniesarebigenough(approximately7-10daysafterthawing).

PassaginghumaniPScellsgrownunderfeeder-freeconditions

1.Useamicroscopetoidentifyregionsofdifferentiation.Markthedifferentiatedcoloniesusinglensmarkeronthebottomoftheplate.

2.RemoveregionsofdifferentiationbyscrapingwithaPipettetiporbyaspiration.

3.AspiratemediumfromthehumaniPScellcultureandrinsewithDPBS(2mL/well).

4.Add0.5mLperwellofaccutase(Cat.no.SCR005,Millipore,diluted1:1withDPBSbeforeuse).Letitstandatroomtemperaturefor1minute.

5.Removeaccutase,andgentlyrinseeachwell2-3timeswith2mLofDMEM/F-12perwelltowashawayremainingenzymes.

6.Add2mL/wellmTeSR1andscrapecoloniesoffwithacellscraper.

7.Transferthedetachedcellaggregatestoa15mLconicaltubeandrinsethewellwithanadditional2mLmTeSR1tocollectanyremainingaggregates.Addtherinsetothe15mLtube.

8.Centrifugethe15mLtubecontainingtheaggregatesat200xgfor5minutesatroomtemperature.

9.Aspiratethesupernatant.ResuspendpelletinmTeSR1containing10µMROCKinhibitorbygentlypipettingandensurethatcellsaremaintainedasaggregates.

10.PlatethehumaniPScellaggregateswithmTeSR1ontoanewplatecoatedwithmatrigel.(Removematrigelsolutionbeforeplating).Ifthecoloniesareatanoptimaldensity,thecellscanbesplitevery5-7daysusing1:3to1:6ratio.

11.Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO2and95%humidity.

12.Changemediumdaily.

CryopreservinghumaniPScells

1.PrepareEZStemfreezingmedium(Cat.no.M050,ALSTEM)onice.

2.Performsteps1-8fromPassaginghumaniPScellsgrownunderfeeder-freeconditions

3.Gentlyaspiratethesupernatantandloosenthecellpelletbytappingthebottomofthetube.

4.Gentlyresuspendthepelletinfreezingmedium,takingcaretoleavetheclumpslargerthanwouldnormallybedoneforpassaging.

5.Transfer1mLofclumpsinfreezingmediumintoeachlabeledcryogenicvial.

6.Placevialsintoanisopropanolfreezingcontainerandplacethecontainerat-80°Covernight.

7.Transfertoaliquidnitrogentanknextday.

Ⅱ.Feeder-dependentcultureconditions

PreparationofhumanESmedium

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

ThawingcryopreservedhumaniPScells

Toinsurethehighestlevelofviability,besuretowarmmediumto37°Cbeforeusingitonthecells.DuetothelowsurvivalrateofcryopreservedhumaniPScells,therecoveryisexpectedtotakeatleastoneweek.

1.QuicklythawthehumaniPScellsina37°Cwaterbathbygentlyshakingthecryovialcontinuouslyuntilhalfthawed.Removethecryovialfromthewaterbathandspraywith70%ethanoltosterilize.

2.Transferthecontentsofthecryovialtoa15mLconicaltube.Add5mLwarmhumanESmediumdropwisetothetube,gentlymixingasthemediumisadded.

3.Centrifugecellsat200xgfor5minutesatroomtemperature.

4.Whilecentrifuging,removeMEFmediumfromthefeedercellplates,andwashthewellstwicewithKnockoutDMEM/F12.Thenadd1mlofhumanESMediumwith10µMROCKinhibitor(Y-27632,StemRD)toonewellof6-wellplate.

5.Aftercentrifugation,aspiratethemediumfrom15mLtube.Gentlyresuspendthecellpelletin1mLfreshhumanESmediumcontaining10µMROCKinhibitor(Y-27632),takingcaretomaintainthecellsassmallcellclumps.

6.Transferthemediumcontainingthecellclumpstoonewellof6-wellplatewithMEFfeedercells.

7.Placetheplateintothe37°Cincubatorandmovetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributetheclumpswithinthewells.Culturethecellsat37°C,with5%CO2and95%humidity.

8.Changemediumdaily.Checkforundifferentiatedcoloniesthatarereadytopassagewhencoloniesarebigenough(approximately7-10daysafterthawing).

PassaginghumaniPScellsgrownunderfeeder-dependentconditions

1.Aspiratethemediumandwashthecellstwicewith1mlofPBS.

2.RemovePBScompletelyandadd0.5mlofAccutase(Cat.no.SCR005,Millipore,diluted1:1withDPBSbeforeuse)andincubatefor1-2minatroomtemperature.

3.Tapthebottomoftheplatetodislodgethecellsfromthebottomoftheplate.Thenaspiratetheaccutase.

4.Add1mlofDMEM/F12totheplateandcarefullywashthefeedercells,andaspiratethemedium.Repeat.

5.Add1mlofhumanESmediumcontaining10µMROCKinhibitortotheplateandsuspendthecellcoloniesbypipettingupanddown.Itisimportantnottobreakupthecoloniesintosinglecells.

6.RemoveaplateofMEFfeedercellsfromtheincubator.AspiratetheMEFmedium.WashoncewithKODMEM/F12medium.

7.Distribute0.2–0.3mlofthehumaniPScellsuspensiontoeachwellofa6-wellplate.AddhumanESmediumwithROCKinhibitortoafinalvolumeof2mlperwell.RightafterplatingtheiPScells,gentlyswirltheplateback-and-forthandside-to-sideandincubateat37°C.

8.After24hours,removethemediaandreplacewithhumanESmedia(withoutROCKinhibitor).

9.ThehumanESmediamustbechangedeverydayandhumaniPScellssubculturedevery5-7days.Trackthepassagenumberofthecells.

CryopreservinghumaniPScells

1.PrepareEZStemfreezingmedium(Cat.no.M050,ALSTEM)onice.

2.Performsteps1-6fromPassaginghumaniPScellsgrownunderfeeder-dependentconditions

3.Gentlyaspiratethesupernatantandloosenthecellpelletbytappingthebottomofthetube.

4.Gentlyresuspendthepelletinfreezingmedium,takingcaretoleavetheclumpslargerthanwouldnormallybedoneforpassaging.

5.Transfer1mLofclumpsinfreezingmediumintoeachlabeledcryogenicvial.

6.Placevialsintoanisopropanolfreezingcontainerandplacethecontainerat-80°Covernight.

7.Transfertoaliquidnitrogentanknextday.

Alstembio原代细胞通常在培养中只能经历有限数量的细胞分裂，然后进入复制衰老，在那里它们不再分裂。科学家需要经常从组织中重建新鲜的培养物，这可能是乏味的，并且会增加从一种制剂到另一种制剂的变异性。来源于原代细胞的永生化细胞可以超过正常的细胞衰老并具有扩展的复制能力。永生化细胞更易于培养和维护，因此对细胞生物学的研究非常有用，从而使科学家能够通过研究项目使用相同的一致细胞进行纵向研究。存在几种在培养条件下使哺乳动物细胞永生化的方法。猿猴病毒40（SV40）T抗原可以在受感染的细胞中诱导端粒酶活性，并且已被证明是使许多不同细胞类型永生化的最简单，最可靠的药物。细胞永生化的最新发现方法是通过表达端粒酶逆转录酶蛋白（TERT）。这种方法对于受端粒长度影响最大的细胞（包括许多人类细胞类型）特别有用。在大多数体细胞中，TERT通常是沉默的。这些细胞可以通过在外源引入hTERT时保持足够的端粒长度来避免复制性衰老。但是，在某些细胞类型（特别是在上皮细胞中）中hTERT的过表达不能诱导细胞永生化。Alstem同时提供SV40和hTERT细胞永生化套件，请参阅下面的订购信息。

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