1.1.CantheNCISKitbeusedonadults?
TheNCISkitisspecificallypackagedforfiveseparatetissuedissociations,eachcontaininguptotwelveneonatalrathearts.TheNCISprotocolisavailableonlineatwww.Worthington-biochem.com/NCIS/default.html.WehaveamodifiedadultratcardiomyocyteisolationtutorialthatwecanfaxuponrequestwhichrecommendstheLangendorfPerfusionSystemandCollagenaseTypeII.1.2.WhatarethevolumesofHank"s,Earle"sandLeibovitzL-15Mediathatyousell?
SterilecalciumandmagnesiumfreeHank"sBalancedSaltSolution,p7.4issuppliedintheNCIS(NeonatalCardiomyocyteIsolationSystem)kit.A500mlbottlemaybepurchased,catalog#LK003210is$42.TheNCISkitalsocontainsapouchofLeibovitzL-15MediaPowder.Reconstitutetheentirecontentsofthepouchintoabeakercontaining800mlofcellculturegradewater.Rinsepouce2-3timeswithadditional100ml.Bringtotalvolumeto1literandfilterthrougha0.22micronporesizefilter.ThepouchcontainingLeibovitzL-15Mediapowderfor1liter,catalog#LK003250is$18.SterileEarle"sBalancedSaltSolution(EBSS)withcalcium,magnesium,bicarbonateandphenolredissuppliedwiththePDS(PapainDissociationSystem)kit.A100mlbottlemaybepurchased,catalog#LK003182is$40.2.1.1.WhatisanacceptableenzymediluentforCollagenase(CLS)?
Collagenasemaybedirectlydissolvedinabalancedsaltsolutionorbufferofchoice.Itshouldbesterilefilteredthrougha0.22umporesizemembrane.ItsoptimumpHrangeis7-9.Calciumisrequired.MostbalancedsaltsolutionshaveenoughCa.Collagenaseisinhibitedbymetalchelatingagentslikecysteine,EDTAoro-phenanthroline.Forourassaywedissolvein0.05MTESwith0.36mMCalciumChloridepH7.52.1.2.WhatisanacceptableenzymediluentforDispase(DISP)?
Dispasecanbedissolvedinwater,balancedsaltsolutionorbufferofchoice.Calciumisanactivatorsimilartocollagenase.2.1.3.WhatareacceptableenzymediluentsforDeoxyribonuclease(D/DPFF/DPRF)?
D/DPFF:Maybedissolvedin5mMacetate,1mMcalcium,pH4.5andstoredinsingleusealiquotsat-20or-70for1yr.DPRF:UsewateroranybufferpH4-9exceptphosphate,avoidcalciumchelators,add50%glycerolforstorageasliquidat-20C.Aliquotavoidrepeatedfreeze/thawcycles.Specialcaremustbetakenwithdeoxyribonuclease.Mixgentlytoavoidsheardenaturation.Ingeneral,DnaseIisactivatedbybivalentmetals.MaximumactivationisattainedwithMg+andCa+.DnaseIisinhibitedbyEDTAandSDS.TheoptimumpHofDeoxyribonucleaseIfromBovinePancreasis7.8.2.1.4.WhatisanacceptableenzymediluentforElastase(ES)?
ElastasehaspoorsolubilityatneutralpHandconcentrationsgreaterthan0.25%.ItishelpfultomakeprimarysolutionsinKCloralkalinebuffersbeforedilutingintoreactionmixturesormedia,compensatingforionicstrengthorpHchanges.ElastaseisstableatpH4.0-10.4,optimumpH8.5.ElastaseisunstableatpHbelow3.5.Elastaseshouldbesterilefilteredthrougha0/.22umporesizemembranebeforeuse.DONOTFREEZE.2.1.5.WhatareacceptableenzymediluentsforPapain(PAP/PAP2)?
Theenzymeshouldbefullyactivatedbeforeuseinasolutioncontaining1.1mMEDTA,0.067mMmercaptoethanoland5.5mMcysteine-HCLfor30minutes.Papainisactivatedbycysteine,sulfideandsulfite.StABIlizingagentsareEDTA,cysteineanddimercaptoethanol.DonotfreezeaqueoussUSPensions.OptimumpHrangeis6.0-7.0.2.1.6.WhatisanacceptableenzymediluentforTrypsin(TRL)?
Stocksolutionsoftrypsinshouldbemadeinitiallybyreconstitutingin0.001NHCL.Thissolutioncabedilutedinthedigestionmediumorbufferimmediatelypriortouse.TheoptimumpHofTrypsinisapproximately8.0.2.2.1.WhatareconversionfactorsforunitsofCollagenase(CLS)?
UnfortunatelythereisnoconsistentconversionunitbetweenWunschandMandlunits.WorthingtonassayisbasedonmodifiedMandlunitapprox.0.15Wunschunitsequal200Mandlunits.2.2.2.WhatareconversionfactorsforunitsofDeoxyribonuclease(D/DP/DPRFF)?
Note:Kunitzunitsasreportedbyothersupplierscanbe2-4timeshigherthanKunitzunitsasmeasuredatWorthington.AtWorthington0.005Kunitzunitdigest1ugoflamBDaDNAin10minat37Cin50mMTris,1mMMg++,pH7.8ina50ulreaction.CorrelationofdigestionunitswithKunitzunitsisdifferentforotherDNAandbuffersystems.Whenproteaseispresent,unitscannotbecorrelated.2.2.3.WhatareconversionfactorsforunitsofTrypsin(TRL)?
1mgTRL>180TAMEunits>10,350BAEEunits>3,450USP/NFunits2.3.Whatistheenzymedigestionscale?
2.4.1.Whatarethebasicdifferencesamongthefourtypesofcollagenase?
CLSI Containsaverageamountsofassayedactivities(collagenase,caseinase,trypticandclostripain).Recommendedforepithelial,liver(hepatocytes),lung,fat(ADIpocytes)andadrenalcellisolations. CLS2 Containsgreaterproteolyticactivities,especiallyclostripainactivities.Itisgenerallyusedforheart(cardiomyocytes),bone,muscle,thyroid,cartilageandendothelialcells. CLS3 Allactivitieslowerthanaverage.Lowcaseinaseandlowproteolyticactivity.Itisusuallyusedformammarycellsandothersofttissues. CLS4 Selectedbecauseoflowtrypticactivity.Highcollagenaserequired.Itiscommonlyusedforpancreaticisletsandotherapplicationswherereceptorintegrityiscrucial. 2.4.2.Whatarethetestspecificationsforeachtypeofcollagenase?
collagenase caseinase clostripain tryptic A280 CLS1 ≥125u/mg ≥200u/mg ≥4.0u/mg ≥0.5u/mg report CLS2 ≥125u/mg ≥200u/mg ≥3.5u/mg ≥0.1u/mg report CLS3 ≥100u/mg ≥50u/mg ≥3.0u/mg ≥0.3u/mg report CLS4 ≥160u/mg ≥100u/mg ≥3.0u/mg ≥0.1u/mg report CLSPA ≥500u/mg ≥50u/mg ≥2.0u/mg ≥0.25u/mg report
