TheWorthingtonPapainDissociationSystemisasetofreagentsintendedforuseintheneuralcellisolationmethodofHuettnerandBaughman(J.Neurosci.,6,3044,1986).Thematerialsaredesignedforconvenienceandsimplicityandareusefultotheoccasionaluseraswellasthemoreexperiencedandfrequentuser.Eachlotisusetestedforperformanceinratspinalneuralcellisolationandthiskitprovidesfreshlypreparedenzymesolutionsforeachdissociation.
Stability/Storage:Thereagentsarestableatambienttemperaturesfortheperiodsoftimeexpectedinnormalshippingprocedures,butthepackageshouldberefrigerateduponarrival.Contentsmaybestoredat2-8°Cfor4-6monthsbeforeuse.Storeat2-8°C.
PackageContents
Thepackagecontainssufficientmaterialsfordissociationoffiveseparatetissuealiquotsofupto0.3-0.4cm3each.Forlargertissuesamplesprepareproportionatelylargervolumesofreagentsateachstepandcombinetheminthesameratioasdescribedintheprotocol.
•Vial1:SterileEarlesBalancedSaltSolution(EBSS)withcalcium,magnesium,bicarbonateandphenolred,onevialperpackage,100ml.Aliquotsofthisvialareusedtoreconstituteothervialsandtopreparediluteinhibitorsolution.RefrigeratebetweenusesandequilibratewithsterileO2:CO2beforeeachuse.
•Vial2:PapaincontainingL-cysteineandEDTA,5x100unitsingle-usevialsperpackage.Thematerialis0.22micronmembranefilteredandlyophilizedinautoclavedvials.Avialreconstitutedwith5mlofEBSS(Vial1)yieldsasolutionat20unitsofpapainpermlin1mML-cysteinewith0.5mMEDTA.Briefincubationat37°Cisneededtoinsurefullsolubilityandactivity.
•Vial3:DeoxyribonucleaseI(DNase),5x2000unitsingleusevialsperpackage.
Thismaterialis0.22micronmembranefilteredandlyophilizedinautoclavedvials.Avialreconstitutedwith0.5mlofEBSS(Vial1)yieldsasolutionat2000unitsofdeoxyribonucleaseperml.Avoidvigorousmixing.
•Vial4:Ovomucoidproteaseinhibitorwithbovineserumalbumin,onevialperpackage,32mluponreconstitution.
Thismaterialis0.22micronmembranefilteredandlyophilizedinautoclavedvials.Avialreconstitutedwith32mlofEBSS(Vial1)yieldsasolutionataneffectiveconcentrationof10mgsofovomucoidinhibitorand10mgsofalbuminperml.Aliquotsofthisvialareusedforeachdissociation.RefrigeratebetweenusesandequilibratewithsterileO2:CO2beforeeachuse.Stableafterreconstitutionwhenstoredat2-8°C.
AlsoincludedisacardcorrelatingcolorwithpHforuseasaguideinO2:CO2equilibration.
RelatedProducts:
Collagenase(CLS1-4/CLSPA)
DeoxyribonucleaseI(DP/D/DCLS/D2/DPFF/DPRF)
Hyaluronidase(HSE/HSEP)
NeutralProtease(Dispase,NPRO)
ProteinaseK(PROK)
Trypsin(TL/TRL/TRL3/TRLS/TRTPCK)
TrypsinInhibitors(LBI/OI/SI/SIC)
CellIsolationOptimizimgSystem(CIT)
HepatocyteIsolationSystem(HIS)
NeonatalCardiomyocyteIsolationSystem(NCIS)
