Alstembio/MouseipCellline/iPS02M-免疫在线蚂蚁淘旗下平台

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Alstembio/MouseipCellline/iPS02M

商品编号： iPS02M

品牌： ALSTEM

市场价： ¥11900.00

美元价： 7140.00

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

MouseiPSCellLine:#iPS02M

ProductDescription

MouseiPScellline(inducedpluripotentstemcellline)wasderivedfrommouseembryonicfibroblasts(MEFs)byretroviralexpressionofOct3/4,Sox2,Klf4andc-Mycgenes.ThecellswerederivedusingmorphologicalselectioncriteriaandwithouttheuseoffluorescentMarkerordrugselection.WhenculturedunderstandardmouseEScellcultureconditions,themorphologyofmouseiPSCsareidenticaltothatofmouseEScells.ThecellsalsoexpressthepluripotencymarkersSSEA-1andNanog,anddemonstratestrongendogenousalkalinephosphataseactivity.MouseiPScellsaregrownonafeederlayerofmouseembryonicfibroblasts(MEFs)andrequirethepretreatmentoftheplatewithGelatin.

ProductSpecifications
ProductName	MouseiPSCellLine
Catalog#	iPS02M
Size	2x10⁵cells/vial
Shipping	DryIce
StorageandStABIlity	Storeingasphaseofliquidnitrogenimmediatelyuponreceipt.Thisproductisstablefor6monthswhenstoredasdirected.
QualityControl	MouseiPScellsweregrowninmouseESmediumsupplementedwith10³U/mlLIF.EachlotofmouseiPScellsistestedforgrowthandviabilityfollowingrecoveryfromcryopreservation.Inaddition,eachlotistestedforexpressionofSSEA-1andNanog,aswellastheactivityofalkalinephosphatase.
SafetyPrecaution	ALSTEMhighlyrecommendsthatprotectivegloves,alabcoat,andafull-facemaskalwaysbewornwhenhandlingfrozenvials.Itisimportanttonotethatsomeliquidnitrogencanleakintothevialswhensubmersedinliquidnitrogen.Uponthawing,theliquidnitrogenreturnstothegasphase,resultinginexcessivepressurewithinthevialthatcancausethevialtoexplodeorexpelthecapwithdangerousforce.
RestrictedUse	ForResearchUseOnly.Notforuseindiagnosticortherapeuticprocedures.
Protocol	Download

Overview
Procedure

ThisprotocolcanbeusedforculturingmouseiPScells.HumaniPScellsweregeneratedbytransducingsourcecellswithretrovirusesindividuallyencodingthefourmousetranscriptionfactors(Oct4,Sox2,Klf4,andc-Myc)thathavebeenshowntoinducethereprogrammingofmouseembryonicfibroblastsintoapluripotentstate.Thecellswerederivedusingmorphologicalselectioncriteriaandwithouttheuseoffluorescentmarkersordrugselection.WhenculturedunderstandardmouseEScellcultureconditions,themorphologyofmouseiPScellsisidenticaltothatofmouseEScells.ThecellsalsoexpressthepluripotencymarkersSSEA-1andNanog,anddemonstrateastrongendogenousAPactivity.

Preparationofculturemedium

MEFmediumDMEMcontaining10%FBS,2mMglutamine,1x10^-4Mnonessentialaminoacids,and50Uand50mg/mlpenicillinandstreptomycin.

MouseESmediumKO-DMEMcontaining15%ES-FBS,2mMglutamine,1x10^-4Mnonessentialaminoacids,1x10^-4M2-mercaptoethanol,1x10³U/mlLIF,and50Uand50mg/mlpenicillinandstreptomycin.

2XFreezingmedium20%DMSOplus80%FBS

Crowthconditionformousefibroblasts

Gelatintreatmentofplates

1.Addenoughsterile/autoclaved0.1%gelatintocoverthebottomofthewells.

Approximateamounts:

Plate/dish	Amount/Well
96well	100μl
48well	300μl
24well	0.5ml
12well	1.0ml
6well	1.5-2.0ml
30mm	1.5-2.0ml
60mm	3.0ml
100mm	4.0-5.0ml

2.Incubatethegelatin-coateddishesforatleast15minat37°C.

3.Aspirateexcessgelatinsolutionbeforeusing.

ThawingMEFcells

Toinsurethehighestlevelofviability,besuretowarmmediumto37°Cbeforeusingitonthecells.Cellsshouldbeplatedataminimumcelldensityof1x10⁴cells/cm².

1.Removethevialfromliquidnitrogenandthawquicklyin37°Cwaterbath.

2.Removethevialfromthewaterbathassoonasthecellsarehalfwaythawed,andsterilizebysprayingwith70%ethanol.

3.Transferthecellswith10mlofMEFmediumtoa15-cmconicaltubeandpelletthecellsbycentrifugationat200xgfor5min.

4.DiscardthesupernatantandresUSPendthecellswith10mlfreshMEFmediumandplatethecellsatseeddensityof1x10⁴cells/cm².

5.Incubateat37°Cwith5%CO₂inairatmosphere,untilthecellsreach80-90%confluency.

6.ChangemediatwiceaweekorwhenpHdecreases.

PassageofMEFcells

Cellsshouldbesplitwhentheyreachconfluency.Asplitbasedonseeddensityof0.5x10⁴cells/cm²isrecommended.

1.DiscardthemediumandwashthecellstwicewithPBS.

2.AspiratePBS,andadd1mlperT75flaskof0.05%trypsin-EDTA,andincubatefor2min.

3.Add5mlofMEFmedium,andbreakupthecellclumpsbygentlypipettingupanddownseveraltimes.

4.Transfercellsintoaconicaltubeandcentrifugeat200gfor5min.

5.Discardthesupernatant,andresuspendthecellpelletin10mlMEFmedium.

6.Countthenumberofcells,platecellsat0.5x10⁴cells/cm²,andincubateat37°Cwith5%CO₂.

FreezingMEFcells

1.Followsteps1-4fromthePassageofCellsabove.

2.Discardthesupernatant,andresuspendthepelletinMEFmedium.Addapproximately1mlforeachT75flask.

3.Countthenumberofcellsanddilutethecellsuspensionto1x10⁷cells/ml.

4.Addanequalvolumeofcold2XFreezingMedia(containing20%DMSOand80%FBS)tothecellsuspension.

5.Aliquot1mlofsuspensionintoeachcryovial(5x10⁶cells/vial).

6.Placevialsintoanisopropanolfreezingcontainerandplacethecontainerat-80°Covernight.

7.Transferthevialstoaliquidnitrogentankforlong-termstorage.

MitomycinCtreatmentofMEF

Atconfluence,MEFcellsaretreatedwithmitomycinCtohaltthedivisionofthecellswhentheyarestillabletoconditionthemediumasthefeederlayersforiPSorEScells.

1.Add6mLoffreshMEFmediumcontaining50μlofmitomycinCsolution(1mg/ml)tooneT75flaskofconfluentMEFcells,andswirlitbriefly.

2.Incubateat37°Cforatleast3h.

3.Afterincubation,aspiratethemitomycinC-containingmediumoffthecells,andwashthecellstwicewith10mlofPBS.

4.AspirateoffPBS,add1mlof0.05%trypsin-EDTA,swirltocovertheentiresurface,andincubatefor2minatroomtemperature.

5.Add5mlofMEFmedium,andbreakupthecellstoasinglecellsuspensionbypipettingupanddown.Countthenumberofcells.Seedthecellsongelatin-coateddishes(1x10⁶cellsper100-mmdish,or1.5x10⁵cellsperwellof6-wellplate).

6.Cellsshouldbereadytousebythenextday.

CultureconditionformouseiPScells

ThawingmouseiPScells

Toinsurethehighestlevelofviability,besuretowarmmediumto37°Cbeforeusingitonthecells.

1.Removethevialfromliquidnitrogenandthawquicklyin37°Cwaterbath.

2.Removethevialfromthewaterbathassoonasthecellsarehalfwaythawed,andsterilizebysprayingwith70%ethanol.

3.Transferthecellswith10mlofmouseESmediumtoa15-cmconicaltubeandpelletthecellsbycentrifugationat200Xgfor5min.

4.Discardthesupernatant,resuspendthecellswithfreshmouseESmedium,andplatethecellsinthewellsof6-wellplatewithMEFfeedercells.

5.Incubateat37°Cwith5%CO_{2_{inairatmosphere,untilthecellsreach80%confluency.}}

6.ChangethemediumeverydayorwhenpHdecreases.

MaintenanceofmouseiPScells

ItisimportanttonotethatdoNOTkeepmouseiPScellsincultureforlongperiodsinordertomaintainthepluripotency.

1.Aspiratethemedium,andwashthecellstwicewith1mlofPBS.

2.RemovePBScompletely,add0.5mlof0.25%trypsin-EDTAsolution,andincubateat37°Cfor2min.

3.Whileincubating,removea6-wellplatewithmitomycinC-treatedMEFsfromtheincubator.AspirateMEFmediumandadd2mlofmouseESmediumtoeachwell.

4.RemovetheplatecontainingmouseiPScellsfromtheincubatorandswirltodislodgethecellsfromthebottomoftheplate.

5.Add1mlofESmediumtotheplateandsuspendthecellsbypipettingupanddowntosinglecellsuspension.

6.Distribute0.2mlofthemouseiPScellsuspensiontoeachwellofthe6-wellplate.RightafterplatingiPScells,gentlyswirltheplateback-and-forthandside-to-sideandincubateat37°C.TheESmediamustbechangedeverydayandmouseiPScellssub-culturedevery2-3d.TrackpassagenumberofiPScells.

FreezingmouseiPScells

1.Growcellstotheexponentialphaseina6-wellplate.

2.Aspiratethemedium,andwashthecellstwicewith2mlofPBS.

3.Add0.5mlof0.25%trypsin-EDTAandincubate2minat37°C.

4.Add2mlofmouseESmedium,andsuspendthecellsbypipettingupanddowntosinglecellsuspension.

5.Transferthecellsuspensiontoa15-mlconicaltube,countthenumberofcellsandspinthecellsat200xgfor5min.

6.Discardthesupernatant,andresuspendthecellswithmouseESmediumtotheconcentrationat1x10⁶cellsperml.

7.Addequalvolumeof2Xfreezingmedium(20%DMSOand80%FBS),andaliquotitat0.5mlpervial.

8.Putthevialsinacell-freezingcontainer,andstorethevialsat–80°Covernight.

9.Transferthevialstoliquidnitrogenforlong-termstorage.

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；

公司介绍

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细胞系慢病毒表达载体 Cell Immortalization Kits iPSC工具包病毒产生慢病毒报告质粒转染试剂 Virus Production Reagents 抗体

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