RatBoneMarrowProgenitorEndothelialCells-Customorder 

CatalogNo. RA-6031

SuggestedMedium: M1168 CompleteMouseEndothelialCellMedium /wKit–500ml

ProductDescription

RatBoneMarrowProgenitorCellsarederivedfromthetibiasandfemursof1-weekoldSpragueDewleyRat.RatBoneMarrowProgenitorCellsaregrowninT25tissuecultureflaskspre-coatedwithgelatin-basedsolutionfor2minandincubatedin CellBIOLOGics’ CultureCompleteGrowthMediumgenerallyfor3-7days.Culturesarethenexpanded.Priortoshipping,cellsaredetachedfromflasksandimmediatelycryo-preservedinvials.Eachvialcontainsatleast1x10⁶ cellspermlandaredeliveredfrozen.

ProductTesting

RatPrimaryBoneMarrow-DerivedEndothelialCellsaretestedforexpressionofMarkersusingantibody,PECAM-1Antibody(M-20,sc-1506,SantaCruz)orZO-1RabbitPolyclonalAntibody(CatalogNo.617300,LifeTechnologies)byimmunofluorescencestaining. ThecellsweretestedforuptakeofDil-Ac-LDL,afunctionalmarkerforendothelialcells. Rat BoneMarrowProgenitorCellsarenegativeforbacteria,yeast,fungiandmycoplasma,andcanbeexpandedfor3-6passagesatasplitratioof1:2underthecellcultureconditionsspecifiedby CellBiologics. Repeatedfreezingandthawingofcellsisnotrecommended.

LaboratoryApplications

Standardbiochemicalproceduresperformedwithprogenitorcellculturesincludecelladhesion,migration,RT-PCR,Westernblotting,immunoprecipitation,immunofluorescentstaining,immunofluorescentflowcytometryorgeneratingcellderivativesfordesiredresearchapplications.

Storageof CellBiologics’ Products

CellBiologics shipsfrozencellsondryice.Onreceipt,immediatelytransferfrozencellstoliquidnitrogen(-180°C)untilreadyforexperimentaluse. Livecellshipmentisalsoavailableonrequest.

AuthorizedUsesof CellBiologics’ Products

Rat BoneMarrowProgenitorCellsfrom CellBiologics aredistributedforresearchpurposesonly.Ourproductsarenotauthorizedforhumanuse,forinvitrodiagnosticproceduresorfortherapeuticprocedures.  Transferorresaleofany CellBiologics’ cellsorproductsfromthepurchasertoothermarkets,organizationsorindividualsisprohibitedby CellBiologics withoutthecompany’swrittenconsent. CellBiologics’ TermsandConditionsmustbeacceptedbeforesubmittinganorder.

Disclaimer

Although Rat BoneMarrowProgenitorCellsareisolatedfromlaboratorymicetestingpathogen-free,investigatorsshouldhandlethecellsthattheyreceivefrom CellBiologics withcautionandtreatallanimalcellsaspotentialpathogens,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:

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

Coatingofflasksordishes:

CoatsterileculturedishesorflaskswithGelatin-BasedCoatingSolution(CellBiologics,CatalogNo.6950)for2minandthenaspiratetheexcesssolutionbeforeseedingcells.

Cellrecoveryfromcryovial:

• Quicklythawcellsincryo-vialbyincubatingthemina37°Cwaterbathfor<1minuntilthereisjustasmallbitoficeleftinthevial.
• Promptlyremovethevialandwipeitdownwith70%ethanol.
• Transfercellsfromthevialtoasterilecentrifugetube.Add8-10mlofpre-warmed CellBiologics CellCultureMedium.Flushthevialwithanadditional0.5-1mlofmediumtoensurecompletetransferofcellstothecentrifugetube.
• Centrifugecellsat200gfor5minutes.
• AspiratethesupernatantandresUSPendthecellpelletin6mlof CellBiologics’ CellCultureGrowthMedium.
• AddresuspendedcellsintoaT25flaskpre-coatedwithGelatin-BasedCoatingSolution(CellBiologics,CatalogNo.6950).
• PlacetheT25flaskinahumidified,5%-CO₂ incubatorat37°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)solutionfor1minute.Use2.0mlof0.05%Trypsin-EDTAsolutionwhencollectingcellsfromaT75flask,and1.0mlwhenusingaT25flask.Assoonascellshavedetached(theflaskmayrequireafewfirmgentletaps),add8-10mlof CellBiologics CellCultureMediumsupplementedwith5-10%FBStoaT25orT75flask(theFBSwillneutralizethetrypsin).
• PlatecellsinfreshflasksorplatesprecoatedwithGelatin-BasedCoatingSolutioninahumidified,5%-CO₂ incubatorat37°C. Changemedium3-6hoursafterseedingcellstoensure>90%cellsareattached. Alsochangeculturemediathefollowingdaytoremovenon-adherentcellsandreplenishnutrients.
• Cellsshouldbecheckeddailyunderamicroscopytoverifyappropriatecellmorphology.
• Changeculturemediumevery24-48hours. Themediumshouldbechangedeverydaywhencellsare>60%confluent toremovenon-adherentcellsandreplenishnutrients.
• Pre-washcellswith1XPBS2timeswheneverreplacingthemedium.

Werecommendsplittingprimarycellsatthefollowratio:

• Therecommendedsplitratioforprimarymurinecellsis1:2.
• AconfluentmonolayerofprimarycellsgrowninaT75flaskmaybeexpandedona6-wellplatereadyforuseinexperimentsunderthecellcultureconditionsspecifiedbyCellBiologics.

ProcedureforFreezingCells

Materials:

• 1XPhosphateBufferedSaline(PBS-1X)
• 0.05%Trypsin-EDTA(1X)solution 
• TissueCultureMedia
• ColdFreezingMedia(10%DMSO,50%FBSand40%culturemedium)
• LabeledCryovials
• Confluentcells

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• Flushtheadherentcelllayerwitha5mlsterilepipette3-5timestodislodgelooselyattachedcells.
• Removeanddiscardthecellculturemediafromtheflask.
• Washadherentcells2-3timeswith10mlofsterilePBS(1X)withoutcalciumandmagnesiumtoremovenonadherentcellsorfraction.
• Removeanddiscardthewashsolutionfromtheflask.
• Incubatecellswithwarm(37°C)Trypsin-EDTA(1X)solutionfor2-5 minute.Use2.0-3.0mlof0.05%Trypsin-EDTAsolutionwhencollectingcellsfromaT75flask,and1.0-1.5mlwhenusingaT25flask.Assoonascellshavedetached(theflaskmayrequireafewfirmgentletaps),add10mlofCellCultureMediumsupplementedwith5-10%FBStotheflask(theFBSwillneutralizethetrypsin).
• Centrifugethecellsuspensionat200gfor5minutes.
• RemovesupernatantwithsterilePasteurpipette.
• Quicklyre-suspendpelletbyadding1.0mlfreezingmediapervialtobefrozen.
• PlacevialsinNalgene"Mr.Frosty"freezingcontainercontaining100%isopropylalcoholat-70-80°Cfor24h.
• TransfervialstoliquidN₂ tankforindefinitestorage.

Werecommendfreezingprimarycellsatthefollowratio:

• AconfluentprimaryendothelialcellsgrowninaT75flaskmaybefrozenin2cryovials.
• AconfluentprimaryendothelialcellsgrowninaT25flaskmaybefrozenin1cryovial.