Alstembio/HumanNeuralStemCellLine/hNSC11,Alstembio,,Alstembio,ALSTEM,+,,Array.Array.Array蚂蚁淘厂家直采.

产品名称：Alstembio/HumanNeuralStemCellLine/hNSC11

产品编号：hNSC11

市场价：¥13900.00

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：$695.00

品牌： Alstembio

公司：ALSTEM Inc

公司分类：

商品介绍

NeuralStemCellLine,Human:#hNSC11

ProductDescription

Neuralstemcells(NSCs)areneuralProgenitorcellsthatarecapableofdifferentiatingtodifferentkindsofcellsinthenervoussystemwhenunderdefinedconditions.Takingadvantageoftheirconsistentpropagation,NSCshavegreatpotentialforthestudyofneurogenesis,neurodegenerativediseases,andclinicaltransplantationapplications.Alstem"shumanNeuralStemCellLineisderivedfromhumaninducedpluripotentstemcells,whicharegeneratedfromadultskinfibroblastsusingAlstem’suniquefootprint-freeepisomaliPSCreprogrammingmethod.ThesecellsareabletoproliferateregularlywithoutdifferentiatingtoneurallineagecellswhenculturedinbFGFandEGFcontainingmedium.

NeuronsderivedfromAlstem"sNSCsstainedwithbeta-III-tubulin

hNSCFeatures

Derivedfromfootprint-freehumaniPScells
Homogeneouspopulation
Candifferentiatetoover90%ofneuronsunderdefinedconditions

ProductSpecifications
ProductName	HumanNeuralStemCellLine
Catalog#	hNSC11
Size	1x10⁶cells/vial
Shipping	DryIce
StorageandStABIlity	Storeingasphaseofliquidnitrogenimmediatelyuponreceipt.Thisproductisstablefor6monthswhenstoredasdirected.
QualityControl	EachlotofhNSCsistestedforgrowthandviabilityafterrecoveryfromcryopreservation.Inaddition,eachlotistestedforexpressionofNestintoensureitsundifferentiatingcharacteristics.
SafetyPrecaution	ALSTEMhighlyrecommendsthatprotectivegloves,alabcoat,andafull-facemaskalwaysbewornwhenhandlingfrozenvials.Itisimportanttonotethatsomeliquidnitrogencanleakintothevialswhensubmersedinliquidnitrogen.Uponthawing,theliquidnitrogenreturnstothegasphase,resultinginexcessivepressurewithinthevialthatcancausethevialtoexplodeorexpelthecapwithdangerousforce.
RestrictedUse	ForResearchUseOnly.Notforuseindiagnosticortherapeuticprocedures.
Protocol	Download

Overview
Procedure

Thisprotocolcanbeusedforculturinghumanneuralstemcells.TheneuralstemcellsarederivedfromALSTEM"sfootprint-freeiPScellsandeachlotistestedfortheexpressionofNestintoensureitsundifferentiatingcharacteristics.

Protocol

Preparationofneuralstemcellculturemedium

1.ThawN-2supplement(100X)(Cat.no.17502-048,LifeTechnologies)andB-27serumfreesupplement(50X)(Cat.no.17504-044)overnightat4°C.

2.Addthe5mLofthawedN-2supplement,10mLofthawedB-27supplement,20ng/mlbFGF(Cat.no.bFGF-50,StemRD)and20ng/mlEGF(Cat.no.EGF-50,StemRD)to500mLofDMEM/F12medium(Cat.no.15-090-CV,Cellgro)aseptically,andmixwell.Filterthrougha0.2μm,low-proteinbindingfilter,ifdesired.

3.Aliquotintoappropriateamountaccordingtousageandstorethealiquotsat4°C.

Coatingplateswithpoly-l-ornithineandlaminin

1.Dilutepoly-l-ornithine(Cat.no.P3655,Sigma)oniceusingsteriletissueculture-gradewater.Aliquotthedilutedpoly-l-ornithinandkeepthealiquotsin-80°Cuntiluse

2.Aliquotlaminin(Cat.no.L2020,Sigma)oniceandkeepthealiquotsin-80°Cuntiluse.

3.Add1mLofpoly-l-ornithine(20µg/mL)toonewellofa6-wellplate,andincubateat37°Cfortwohours.

4.Aspiratepoly-l-ornithineandrinsethewelltwicewith1mLofDPBS.Add1mLoflamininsolution(5µg/mL)inthewellandincubateat37°Cfor2hours.

5.Aspiratethelamininbeforeplatingcellsorstoreplateat4°Cuntilneeded.

CoatingplateswithMatrigel(alternativeplatecoatingmethod)

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

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

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

3.Keepthecoatedplateat37°Cfor1hourorovernightat4°C.Ifplatehasbeenstoredat4°C,allowtheplatetoincubateat37°Cforatleast30minutesbeforeremovingthematrigelsolution.

Preparationofneuralstemcellfreezingmedium

1.Add9mLofneuralstemcellculturemediumwith1mLofDMSOina15mLconicaltube.Keepitat4°Cuntiluse.Prepareneuralstemcellfreezingmediumimmediatelybeforeuse.Donotstore.

ThawingcryopreservedhNSC

1.QuicklythawtheNSCsina37°Cwaterbathbygentlyshakingthecryovialcontinuouslyuntilhalfthawed.Removethecryovialfromthewaterbathandspraywith70%ethanoltosterilize.

2.Transferthecontentsofthecryovialtoa15ofmLconicaltube.Add5mLofwarmN2/B27mediumcontainingbFGFandEGFtothetube,gentlymixingasthemediumisadded.

3.Centrifugecellsat200xgfor5minutesatroomtemperature.

4.Whilecentrifuging,removethematrigelsolutionfromacoatedtissueculture6-wellplate.Add1mLofwarmN2/B27mediumcontainingbFGFandEGFtoonewellofthe6-wellplate.

5.Aftercentrifugation,aspiratethemediumfrom15mLtube.GentlyresUSPendthecellpelletin1mLN2/B27mediumcontainingbFGFandEGF,followingbytransferringthecellstothematrigelcoated6-wellplate.

6.Movetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributethecellswithinthewell,andthenplacetheplateintothe37°Cincubator.Culturethecellsat37°C,with5%CO2and95%humidity.

7.Changemediumineverytwodays.

1.Passagecellswhenthecellsreach80-90%confluence.

2.AspiratemediumfromthehNSCcultureandrinsewithDPBS(2mL/well).

3.Add0.5mLperwellofaccutase(Cat.no.SCR005,Millipore).Keeptheplateat37°Cfor2-3minutes.

4.Neutralizetheaccutasewith1mlofN2/B27mediumcontainingbFGFandEGF.Gentlyrinseeachwell2-3timestoliftupmostofthehNSCs.

5.Transferthedetachedcellstoa15mLconicaltubeandrinsethewellwithanadditional2mLofN2/B27containingbFGFandEGFtocollectanyremainingcells.Addtherinsetothe15mLtube.

6.Centrifugethe15mLconicaltubecontainingthecellsat200xgfor5minutesatroomtemperature.

7.Aspiratethesupernatant.ResuspendthepelletinN2/B27containingbFGFandEGFbygentlypipettingupanddown.

8.PlatethehNSCsusing1:6ratioswith2mLN2/B27mediumcontainingbFGFandEGFinanewplatecoatedwithpoly-l-ornithineandlamininormatrigel(Removethecoatingsolutionbeforeplatingthecells).

9.Movetheplateinquicksidetoside,forwardtobackmotionstoevenlydistributethecellswithinthewells,andthenplacetheplateintothe37°Cincubator.Culturethecellsat37°C,with5%CO2and95%humidity.

10.Changemediumoncepertwodays.