Cell Biologics/C57BL/6 Mouse Primary Embryonic Fibroblasts/C57-6028/ Fro

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¥1400.00
货号:C57-6028
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品牌:Cell Biologics
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商品描述

C57BL/6MousePrimaryEmbryonicFibroblasts

CatalogNo.C57-6028

SuggestedMedium:FibroblastMedium/wKit(500ml)

CatalogNo.M2267

ProductDescription

C57BL/6MousePrimaryEmbryonicFibroblastsfromCellBIOLOGicsareisolatedfromtissueofpathogen-freelaboratorymice.MousePrimaryEmbryonicFibroblastsaregrowninT25tissuecultureflaskspre-coatedwithgelatin-basedsolutionfor0.5hourandincubatedinCellBiologicsCultureCompleteGrowthMediumgenerallyfor3-7days.Culturesarethenexpanded. Priortoshipping,cellsaredetachedfromflasksandimmediatelycryo-preservedinvials.Eachvialcontainsatleast2x106cellspermlandisdeliveredfrozen.MousePrimaryEmbryonicFibroblastsarenegativeforbacteria,yeast,fungi,andmycoplasma.CellsaretestedforexpressionofMarkerusingtheantibodyofanti-FSP1/S100A4(MilliporeUSA)byimmunofluorescencestaining.Cellscanbeexpandedfor3-6passagesatasplitratioof1:2underthecellcultureconditionsspecifiedbyCellBiologics.Repeatedfreezingandthawingofcellsisnotrecommended.

LaboratoryApplications

Standardbiochemicalproceduresperformedwithcellculturesincludetheassayofcelltocellinteraction,RT-PCR,Westernblotting,immunoprecipitation,immunofluorescentstaining,flowcytometryorgeneratingcellderivativesfordesiredresearchapplications.

StorageofCellBiologicsProducts 

CellBiologicswillshipfrozencellsondryice.Onreceipt,immediatelytransferfrozencellstoliquidnitrogen(-180°C)untilreadyforexperimentaluse.Live-cellshipmentisalsoavailableonrequest.

AuthorizedUsesofCellBiologics’Products 

MousePrimaryEmbryonicFibroblastsfromCellBiologicsaredistributedforresearchpurposesonly.Ourproductsarenotauthorizedforhumanuse,forinvitrodiagnosticproceduresorfortherapeuticprocedures.TransferorresaleofanyCellBiologics’cellsorproductsfromthepurchasertoothermarkets,organizationsorindividualsisprohibitedbyCellBiologicswithoutthecompany’swrittenconsent.CellBiologics’TermsandConditionsmustbeacceptedbeforesubmittinganorder.

Disclaimer

AlthoughMousePrimaryEmbryonicFibroblastsareisolatedfromlaboratorymicetestingpathogen-free,investigatorsshouldhandlethecellsthattheyreceivefromCellBiologicswithcautionandtreatallanimalcellsaspotentialpathogens,sincenotestprocedurecancompletelyguaranteetheabsenceofinfectiousagents.

WarrantyandLiABIlity

CELLBIOLOGICS’ guarantee applies only toyour purchase ofCELLBIOLOGICS'cellswithCELLBIOLOGICS’MediaandCoatingSolutionforappropriatecellculturewithin35daysfromthedateofproductdelivery.

PrimaryCellCultureProtocol

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Allcellcultureproceduresmustbeconductedinabio-safetycabinet.

Anyandallmedia,supplements,andreagentsmustbesterilizedbyfiltrationthrougha0.2µmfilter.

Useaseptictechniquetopreventmicrobialcontamination.

Cryo-preservedcellsmustbestoredinliquidnitrogenorseededimmediatelyuponarrival.

Medium:

ReviewtheinformationprovidedontheCellBiologicswebsiteaboutappropriateculturemedia(e.g.serumandothersupplements).Usepre-warmed(37°C)cellculturemedia(30-50ML)torecovercryo-preservedcellsandwhenchangingmediaorsplittingcells.

Coatingofflasksordishes:

CoatsterileculturedishesorflaskswithGelatin-BasedCoatingSolution(CellBiologics,CatalogNo.6950)for10-30minandthenaspiratetheexcesssolutionbeforeseedingcells.

Cellrecoveryfromcryovial:

  • Quicklythawcellsincryo-vialbyincubatingthemina37°Cwaterbathfor<1minuntilthereisjustasmallbitoficeleftinthevial.
  • Promptlyremovethevialandwipeitdownwith70%ethanol.
  • Transfercellsfromthevialtoasterilecentrifugetube.Add8-10mlofpre-warmedCellBiologicsCellCultureMedium.Flushthevialwithanadditional0.5-1mlofmediumtoensurecompletetransferofcellstothecentrifugetube.
  • Centrifugecellsat200gfor5minutes.
  • AspiratethesupernatantandresUSPendthecellpelletin6mlofCellBiologics’CellCultureGrowthMedium.
  • AddresuspendedcellsintoaT25flaskpre-coatedwithGelatin-BasedCoatingSolution(CellBiologics,CatalogNo.6950).
  • PlacetheT25flaskinahumidified,5%-CO2incubatorat37°C.Changemedium3-6hoursafterthawingcellstoensure>90%cellsareattached.Alsochangeculturemediathefollowingdaytoremovenon-adherentcellsandreplenishnutrients.
  • Changecellculturemediumeverydaywhencellsare>60%confluent.
  • Cellsshouldbecheckeddailyunderamicroscopetoverifyappropriatecellmorphology.

Expansionofculturedmouseprimarycells:

  • Flushtheadherentlayerwitha5mlsterilePipette3timestodislodgelooselyattachedcells.
  • Removeanddiscardthecellculturemediafromtheflask.
  • Washadherentcells2timeswith10mlofsterilePBS(1X)withoutcalciumandmagnesiumtoremovenonadherentcellsorfraction.
  • Removeanddiscardthewashsolutionfromtheflask.
  • Incubatecellswithwarm(37°C)0.05%Trypsin-EDTA(1X)solution(Invitrogencatalognumber25300)for1minutes.Use2.0mlof0.05%Trypsin-EDTAsolutionwhencollectingcellsfromT75flasks,and1.0mlwhenusingT25flasks.Assoonascellshavedetached(theflaskmayrequireafewfirmgentletaps),add8-10mlofCellBiologicsCellCultureMediumsupplementedwith5-10%FBStoaT25orT75flask(theFBSwillneutralizethetrypsin).
  • PlatecellsinfreshflasksorplatesprecoatedwithGelatin-BasedCoatingSolutioninahumidified,5%-CO2incubatorat37°C.Changemedium3-6hoursafterseedingcellstoensure>90%cellsareattached.Alsochangeculturemediathefollowingdaytoremovenon-adherentcellsandreplenishnutrients.
  • Cellsshouldbecheckeddailyunderamicroscopytoverifyappropriatecellmorphology.
  • Changeculturemediumevery24-48hours.Pleasenotethatthemediumshouldbechangedeverydaywhencellsare>60%confluenttoremovenon-adherentcellsandreplenishnutrients.
  • Pre-washcellswith1XPBS2timeswheneverreplacingthemedium.

Werecommendsplittingprimarycellsatthefollowratio:

  • Therecommendedsplitratioforprimarymurinecellsis1:2or1:3.
  • AconfluentmonolayerofprimarycellsgrowninaT75flaskmaybeexpandedona6-wellplatereadyforuseinexperimentsunderthecellcultureconditionsspecifiedbyCellBiologics.

ProcedureforFreezingCells

Materials:

  • 1XPhosphateBufferedSaline(PBS-1X)
  • 0.05%Trypsin-EDTA(1X)solution(Invitrogencatalognumber25300)
  • TissueCultureMedia
  • ColdFreezingMedia(10%dimethylsulfoxide(DMSO),20%FBSand70%culturemedium)
  • LabeledCryovials
  • Confluentcells

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  • Flushtheadherentcelllayerwitha5mlsterilepipette3-5timestodislodgelooselyattachedcells.
  • Removeanddiscardthecellculturemediafromtheflask.
  • Washadherentcells2-3timeswith10mlofsterilePBS(1X)withoutcalciumandmagnesiumtoRemovenonadherentcellsorfraction.
  • Removeanddiscardthewashsolutionfromtheflask.
  • Incubatecellswithwarm(37°C)Trypsin-EDTA(1X)solutionfor1minutes.Use2.0-3.0mlof0.05%Trypsin-EDTAsolutionwhencollectingcellsfromT75flasks,and1.0-1.5mlwhenusingT25flasks.Assoonascellshavedetached(theflaskmayrequireafewfirmgentletaps),add10mlofCellCultureMediumsupplementedwith5-10%FBStotheflask(theFBSwillneutralizethetrypsin).
  • Centrifugethecellsuspensionat200gfor5minutes.
  • RemovesupernatantwithsterilePasteurpipette.
  • Quicklyre-suspendpelletbyadding1mlfreezingmediapervialtobefrozen.
  • PlacevialsinNalgene"Mr.Frosty"freezingcontainercontaining100%isopropylalcoholat-70-80°Cfor24h.
  • TransfervialstoliquidN2tankforindefinitestorage.

Werecommendfreezingprimarycellsatthefollowratio:

  • AconfluentprimarycellsgrowninaT75flaskmaybefrozenin2or3cryovials.
  • AconfluentprimarycellsgrowninaT25flaskmaybefrozenin1cryovial.
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