RatBoneMarrowMesenchymalStemCells

CatalogNo. RA-6043

SuggestedMedium: M5566 CompleteMesenchymalStemCellMedium(Animal)/wKit–500ml 

ProductDescription

RatBoneMarrowMesenchymalStemCellsarederivedfromthetibiasandfemursof6-weekoldSprague–DawleyRats.RatBoneMarrowMesenchymalStemCellsaregrowninT25tissuecultureflaskspre-coatedwithgelatin-basedsolutionfor2minandincubatedin CellBIOLOGics’CellCultureMediumfor7-15days.Culturesarethenexpanded. Priortoshipping,cellsaredetachedfromflasksandimmediatelycryo-preservedinvials.Eachvialcontainsatleast1x10⁶ cellspermlandisdeliveredfrozen.RatBoneMarrowMesenchymalStemCellsarenegativeforbacteria,yeast,fungi,andmycoplasma.Cellscanbeexpandedonamultiwellcultureplatereadyforexperimentsunderthecellcultureconditionsspecifiedby CellBiologics. Repeatedfreezingandthawingofcellsisnotrecommended.

MouseBoneMarrowMesenchymalStemCellsweretestedforexpressionofMarkersusingantibodies,CD44,Sca-1andCD29byflowcytometry.

LaboratoryApplications

Standardbiochemicalproceduresperformedincludetheassaysofcelltocellinteraction,PCR,Westernblotting,immunoprecipitation,immunofluorescentstaining,immunofluorescentflowcytometryorgeneratingcellderivativesfordesiredresearchapplications.

Storageof CellBiologics Products 

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

AuthorizedUsesof CellBiologics’ Products 

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

Disclaimer

Investigatorsshouldhandlethecellsthattheyreceivefrom CellBiologics withcautionandtreatallanimalcellsaspotentialpathogens,sincenotestprocedurecancompletelyguaranteetheabsenceofinfectiousagents.

WarrantyandLiABIlity

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

PrimaryCellCultureProtocol

>>>>>>>>>>>>>>>>>>>>>>>> 

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. Changemedium3-6hoursafterthawing/seedingcellstoensure>90%cellsareattached. Alsochangeculturemediathefollowingdaytoremovenon-adherentcellsandreplenishnutrients.
• Changecellculturemediumeverydaywhencellsare>60%confluent.
• Cellsshouldbecheckeddailyunderamicroscopetoverifyappropriatecellmorphology.

Expansionofculturedmouseprimarycells:

• Flushtheadherentlayerwitha5mlsterilePipette3timestodislodgelooselyattachedcells.
• Removeanddiscardthecellculturemediafromtheflask.
• Washadherentcells2timeswith10mlofsterilePBS(1X)withoutcalciumandmagnesiumtoremovenonadherentcellsorfraction.
• Removeanddiscardthewashsolutionfromtheflask.
• Incubatecellswithwarm(37°C)0.25%Trypsin-EDTAsolutionfor1minute.Use2.0mlof0.25%Trypsin-EDTAsolutionwhencollectingcellsfromaT75flask,and1.0mlwhenusingaT25flask.Assoonascellshavedetached(theflaskmayrequireafewfirmgentletaps),add8-10mlof CellBiologics CellCultureMediumsupplementedwith5-10%FBStoaT25orT75flask(theFBSwillneutralizethetrypsin).
• PlatecellsinfreshflasksorplatesprecoatedwithGelatin-BasedCoatingSolutioninahumidified,5%-CO₂ incubatorat37°C. Changemedium4-6hoursafterseedingcellstoensure>90%cellsareattached.
• Alsochangeculturemediathefollowingdaytoremovenon-adherentcellsandreplenishnutrients.
• Cellsshouldbecheckeddailyunderamicroscopytoverifyappropriatecellmorphology.
• Changeculturemediumevery24-48hours.
• Mediumshouldbechangedeverydaywhencellsare>60%confluent toremovenon-adherentcellsandreplenishnutrients.
• Pre-washcellswith1XPBS2timeswheneverreplacingthemedium.

Werecommendsplittingprimarycellsatthefollowratio:

• Therecommendedsplitratioforprimarymurinecellsis1:2.
• AconfluentmonolayerofprimarycellsgrowninaT75flaskmaybeexpandedona6-wellplatereadyforuseinexperimentsunderthecellcultureconditionsspecifiedbyCellBiologics.