Worthington/Neonatal Cardiomyocyte Isolation System/LK003230/1 vi

InhibitorVial,NCISSource:SoybeanAcomponentoftheNCISkit.Thismaterialis0.22micronmembranefilteredandlyophilizedinautoclavedvials.Avialreconstitutedwith1mlofHBSSorequivalentyieldsasolutionof2mg/mloftrypsininhibitor,Code:SIC.Suitableforcellisolationandcultureapplications.Storeat2-8°C.N/A

TheWorthingtonNeonatalCardiomyocyteIsolationSystemhasbeendevelopedtoprovideresearcherswithareliable,convenient,andconsistentneonatalratcardiomyocytecellisolationmethod.Byusingpurifiedratherthancrudeenzymepreparations,ithasbeenpossibletominimizethelot-to-lotvariation.Inaddition,Worthingtonuse-teststhekitsbyisolatingcardiomyocytesfromneonatalratheartstoassureperformance,reliabilityandconsistentyieldofviablecells.

ThekithasbeenformulatedincollaborationwithDr.RonalMacGregor.ThemethodisbasedonthatdescribedbyToraason,etal.Toxicol,56,107(1988)inwhichthemincedtissueisincubatedovernightwithpurifiedtrypsinat2-8°C.AspointedoutbyToraason,thisstepreducesthehands-ontimerequiredtoharvestcellscomparedtothetimeinvolvedinsequentialincubationsinwarmtrypsinorCollagenase.Purifiedcollagenaseratherthancrudecollagenaseisusedtomaximizeyieldandviability.

ContentsofKit
Thepackagecontainssufficientmaterialsforfiveseparatetissuedissociations,eachcontaininguptotwelvehearts.Forlargerorsmallertissuesamplesprepareproportionatevolumesofreagentsateachstepandcombinetheminthesameratioasdescribedintheprotocol.

•Vial1:1bottle,500ml:Sterilecalcium-andmagnesium-freeHanksBalancedSaltSolution(CMFHBSS),pH7.4.ThesolutionisusedforreconstitutingthecontentsofVials#2and#3inadditiontoservingasthemediumforthedissociation.

•Vial2:5vials,1000µgeach:WorthingtonTrypsin(Code:TRLS),3Xcrystallized,dialyzedagainst1mMHCl,filteredthrough0.22micronporesizemembrane,andlyophilized.Beforeuse,reconstitutewith2mlCMFHBSS(Vial#1)andswirlgentlytodissolvecontents.Storeat2-8°C.

•Vial3:5vials,2000µgeach:WorthingtonSoybeanTrypsinInhibitor(Code:SIC),a0.22micronporesizemembranefiltered,lyophilizedpowder.Beforeuse,reconstitutewith1mlCMFHBSS(Vial#1)andswirlgentlytodissolvecontents.Storeat2-8°C.

•Vial4:5vials,1500Unitseach:WorthingtonPurifiedCollagenase(Code:CLSPA),a0.22micronporesizemembranefiltered,lyophilizedpowderwhichhasbeenchromatographicallypurified.Itcontainslessthan50caseinaseunitspermilligramandiscomposedoftwoseparablebutverysimilarcollagenases.Beforeuse,reconstitutewith5mlLeibovitzL-15media(preparedasdescribedbelow)andswirlgentlytodissolvecontents.Storeat2-8°C.

•PouchcontainingLeibovitzL-15MediaPowder:1x1L,Reconstituteentirecontentsofpouchbycuttingopentopofenvelopeandpouringcontentsintobeakercontaining800mlofcellculturegradewater.Rinsepouch2-3timeswithadditional100ml.Bringtotalvolumeto1literandfilterthrougha0.22micronporesizefilter.

Thekitalsoincludes5CellStrainers(Falcon),acardcorrelatingphenolredcolorwithpHforcheckingbalancedsaltsolutionsandculturemedia.

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