CyagenBiosciencesInc.www.cyagen.comOriCellTMStrainBalb/CMouseMesenchymalStemCellsCatalogNo.MUCMX-01001InstructionsforUseMaterialsRequired(notsupplied)1.Trypsin-EDTA(Cat.No.TEDTA-10001-100）2.Phosphate-BufferedSaline(1×PBS)(Cat.No.PBS-10001-500)3.Balb/CMouseMesenchymalStemCellGrowthMedium(Cat.No.MUCMX-90011)ThawingofBalb/CMouseMesenchymalStemCellsPrepare37°Cwaterbathandpre-warmBalb/CMouseMesenchymalStemCellGrowthMediumto37°C.1.Add9mLofBalb/CMouseMesenchymalStemCellGrowthMediumtoa15mLconicaltube.2.RemovethecryovialofBalb/CMouseMesenchymalStemCellsfromliquidnitrogen.Quicklythawthevialin37°Cwaterbathuntilthelastcrystalpiecedisappears,andfinishthethawingprocedurewithin3minutes.Becarefulnottosubmergetheentirevial.Maximumcellviabilityisdependentontherapidandcompletethawingoffrozencells.Note:Thawingthecellsforlongerthan3minutesresultsinlessthanoptimalresults.3.Assoonasthecellsarecompletelythawed,disinfecttheoutsideofthevialwith70%ethanol.4.Inalaminarflowhood,usepipettetotransferthecellstotheconicaltubecontainingBalb/CMouseMesenchymalStemCellGrowthMedium.Becarefulnottointroduceanybubblesduringthetransferprocess.5.Rinsethevialwith1mLofmediumtoreducethelossofcellandthentransferthis1mLofcellsuspensiontotheconicaltube.6.Gentlymixthecellsuspensionbyslowlypipetingupanddown.Becarefulnottointroduceanybubbles.7.Centrifugethecellsuspensionat250gfor5minutes.8.Carefullyaspirateasmuchofthesupernatantaspossibleandadd2-3mLoffreshBalb/CMouseMesenchymalStemCellGrowthMedium(pre-warmedto37°C).9.Gentlyre-suspendthecellsinBalb/CMouseMesenchymalStemCellGrowthMedium.10.PlatethecellsintoaT25flaskandaddsufficientBalb/CMouseMesenchymalStemCellGrowthMedium.Gentlyrockthecultureflasktoevenlydistributethecells.CBPI0055A1MUCMX-010011/3CyagenBiosciencesInc.www.cyagen.com11.Incubateat37°Cina5%CO2humidifiedincubator.12.Thenextday,changethemediumwithfreshBalb/CMouseMesenchymalStemCellGrowthMedium(pre-warmedto37°C).13.Changethegrowthmediumeverythreedaysthereafter.14.Whenthecellsareapproximately80to90...
