C57BL/6MousePrimaryEmbryonicFibroblasts

CatalogNo.C57-6028

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.