PapainDissociationSystemSource:N/ASetoffivesingleusevialsofpapainandfivesingleusevialsofDNase,100mlofEarlesbalancedsaltsolution(EBSS),andaninhibitorvialforuseinthetissuedissociationmethodofHuettner,J.E.,andBaughman,R.W.:J.Neuroscience,6,3044(1986).UsetestedbyWorthingtonusingnew-bornratpupspinalcord.Thepackagecontainssufficientmaterialsfordissociationoffiveseparatetissuealiquotsofupto0.3-0.4cm3each.Storeat2-8°C.

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.

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