Alstembio/HumanipCellline（逆转录病毒）/iPS01-免疫在线蚂蚁淘旗下平台

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商品详细Alstembio/HumanipCellline（逆转录病毒）/iPS01

Alstembio/HumanipCellline（逆转录病毒）/iPS01

商品编号： iPS01

品牌： ALSTEM

市场价： ¥17900.00

美元价： 10740.00

产地：美国(厂家直采)

公司：

产品分类：细胞代谢

公司分类： cellular_metabolism

联系Q Q： 3392242852

电话号码： 4000-520-616

电子邮箱： info@ebiomall.com

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商品介绍

HumaniPSCellLine(Retroviral):#iPS01

Description

HumaniPS(inducedpluripotentstem)celllinewasderivedfromhumanforeskinfibroblasts(HFFs)byretroviralexpressionofOCT4,SOX2,KLF4,andc-MYCgenes.ThecellswerederivedusingmorphologicalselectioncriteriaandwithouttheuseoffluorescentMarkerordrugselection.WhenculturedunderstandardhumanEScellcultureconditions,themorphologyofhumaniPScellsareidenticaltothatofhumanEScells.ThecellsalsoexpressthepluripotencymarkersSSEA-4andNanog,anddemonstratestrongendogenousalkalinephosphatase.

ProductSpecifications
ProductName	HumaniPSCellLine(Retroviral)
Catalog#	iPS01
Size	2x10⁵cells/vial
Shipping	DryIce
StorageandStABIlity	Storeingasphaseofliquidnitrogenimmediatelyuponarrival.Thisproductisstablefor6monthswhenstoredasdirected.
QualityControl	HumaniPScellsweregrowninhumanESmediumsupplementedwith10ng/mlofbFGF.EachlotofhumaniPScellsistestedforgrowthandviabilityfollowingrecoveryfromcryopreservation.Inaddition,eachlotistestedforexpressionofSSEA-4andNanog,aswellastheactivityofalkalinephosphatase.
SafetyPrecaution	ALSTEMhighlyrecommendsthatprotectivegloves,alabcoat,andafull-facemaskalwaysbewornwhenhandlingfrozenvials.Itisimportanttonotethatsomeliquidnitrogencanleakintothevialswhensubmersedinliquidnitrogen.Uponthawing,theliquidnitrogenreturnstothegasphase,resultinginexcessivepressurewithinthevialthatcancausethevialtoexplodeorexpelthecapwithdangerousforce.
RestrictedUse	ForResearchUseOnly.Notforuseindiagnosticortherapeuticprocedures.
Protocol	Download

Overview
Procedure

ThisprotocolcanbeusedforculturinghumaniPScells.HumaniPScellsweregeneratedbytransducingsourcecellswithretrovirusesindividuallyencodingthefourhumantranscriptionfactors(Oct4,Sox2,Klf4,andc-Myc)thathavebeenshowntoinducethereprogrammingofsomaticcellstoapluripotentstate.Thecellswerederivedusingmorphologicalselectioncriteriaandwithouttheuseoffluorescentmarkersordrugselection.WhenculturedunderstandardhumanEScellcultureconditions,themorphologyofhumaniPScellsisidenticaltothatofhumanEScells.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.Dilutematrigel1:30to1:50withpre-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.

品牌介绍

AlstembioiPSC一代服务申请表无风险的一站式服务我们高效的定制iPSC生成服务使您摆脱了繁重的单元重编程过程，因此您可以放松身心并享受自己的研究。我们的iPSC生成服务的主要功能：保证：在极少数情况下无法成功进行编程的情况下，ALSTEM对项目总额具有独特的100％退款政策。这使您可以无风险地试用我们的服务。接受多种供体细胞类型：我们已经成功地从成纤维细胞，PBMC，CD34和LCL生成了iPSC；尖端技术：我们提供无馈线，非集成式重新编程；灵活的重编程方法：我们提供附加型和逆转录病毒iPSC重编程；交付物的多种选择： ALSTEM旨在提供5个来自不同菌落的iPSC小瓶（> 2 x 10 ^ 5个细胞/小瓶）；短期： 2-3个月，取决于重新编程的方法，源细胞和种类；iPSC表征：多能性标志物Oct4和TRA-1-60的AP染色和免疫染色；其他“附加”服务：基因组编辑，畸胎瘤形成，胚状体形成，核型分析和进一步鉴定；

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