Liverisaconvenientsourceforfunctionalintactmitochondriaforanumberofreasons.AnimaltissueismorereADIlyhomogenizedthanplanttissuebecausetherearenocellwalls,andliverinparticularisasoftandfairlyhomogeneoustissue.Themetabolismofendothermsrequiresthatsometissuesmaintainahighdensityofmitochondria,sothepotentialyieldishigh.Isolatingmitochondriafromhighlystructuredanimaltissuessuchasmusclecanbetechnicallydifficultsinceahighproportionoftheorganellesremaintrappedincellandtissuefragments(althoughmusclecanbeagoodsource).Onecanquicklyandeasilyobtainasubstantialquantityoflivermitochondriawithinlessthananhour"spreparationtime.

WeuseSprague-Dawleyalbinomalerats(CharlesRiversLaboratories,Wilmington,MA)forourstudies.Femaleratscanbeused,howeverforseriousstudiesweusuallysticktomalestoavoidcomplicationsduetotheestrouscycle.Agoodweightrangefortheanimalis200-250gms,perhapsabitlarger.Aliverfromthissizeanimalyieldsuptotwomlorsoofconcentratedmitochondria,enoughfordozensofoxygraphexperiments.Intheteachinglab,twosetsoflabpartnersshareoneanimal,dividingthelivertissueintoequalstartingamountsbyweight.SincetheliverisresponsIBLefordetoxificationprocessesincludingmetabolismofanestheticagents,andbecausebothananestheticanditsmetabolitescanaffectliverfunction,itispreferablefromanexperimentalviewpointtoavoidusinganestheticortranquilizingdrugs.Wesedatetheanimalsusingisofluranethendecapitatethemusingaratguillotine.Isofluraneisshortactingandhasnotappearedtocompromisemitochondriafunction.

Theadjective"medial"isananatomicaltermmeaningvertical,upthemiddle.Weopenuptheanimalwithamedialincision(nota"medical"incision)fromgrointosternum.Wefirstseparatetheskinthentheunderlyingmuscleandperitoneum,revealingtheliver.Theliverisbrown,large,andalmostunmistakable.Ifthestudentgetsintotheratquickly(openedwithin2-3minofdecapitation)andcutsthroughthesternum,openingthechest,theheartcanbeseenstillbeating.Thelivershouldbechilledimmediatelybypouringagenerousamount(100ml)ofice-cold0.85%NaClintotheperitonealcavity.Itcanberemovedinpiecesorremovedintactbycutingitoffatthebase,andthenitshouldbedroppedintoasecondbeakerofice-coldsalinesolutiontocontinuetoreducethetemperature.Furtherstepsintheisolationshouldallbedoneatice-buckettemperature.

Wefinditconvenienttodividethetissuefromone200-225gmratintotwoequalportions,eachweighing3to5grams.Theratioofhomogenizingmediumtotissueisimportant,asisthedepthofliquidinthecontainerwhenusingashearingtypehomogenizer.Thespecificinstrumentweuseisa"Tissuemizer"withT25stainlesssteelshaft(Tekmar,Inc.,Cincinnatti,OH).Ateflon-on-glass(Potter-Elvehjem)homogenizercanalsobeused,butittakeslonger.Ashearingtypehomogenizerisfastbutprolongedoroverlyvigoroushomogenizationcanbedamagingtomitochondria.Bothmethodsyieldgoodpreparationsfromratliver.Wehavehadgreatsuccessbydrainingthetissuethenmincingitina50mlplasticdisposablebeaker,followedbyadditionof20mlhomogenizingmedium(0.25Msucrose,5mMHEPESbuffer,and1mMEDTA,pH7.2).AftermixingtosUSPendthemincewehomogenizeatasettingof"40"for10sec.

Toremovelargecellandtissuefragmentsandcellnuclei(the"nuclearpellet"),wecentrifugethehomogenateat500xgfor10minutes.Beforecentrifugation,wetopoffthehomogenatewithmediumtofilleachtube.Tobringdownthemitochondrialpelletwepourthesupernatantintoacleancentrifugetube,andwithouttoppingoffwecentrifugeat9400xgfor10min.

Whenthesupernatantispouredoff,thelooseupperpartofthemitochondrialpelletmaycomeoffaswell.Intactmitochondriatendtosedimentmorequicklythandamagedmitochondria.Theloosepartofthepelletmostlikelycontainsahighproportionofdamaged(uncoupled)mitochondria,andcanbelost.Thewhitefoamymaterialnearthetopofthetubeconsistsoflipids,whichmustbekeptfromcontactwiththemitochondria.Theycanberemovedbywipingtheinsideofthetubewithalabwiper.Mixingoflipidswiththemitochondriasuspensionwillcausesomedegreeofuncoupling(lossofABIlitytomaintainrespiratorycontrol).Afterusingapasteurpipettoremovethelastbitofliquid,aglassrodshouldbeusedtostirtheremainingpelletintoasmoothpaste.Wedon"taddbufferatall.Themoreliquidthatremainswiththepellet,themoredifficultitistohomogenizealloftheparticles.Wekeepthecentrifugetubeonicewhilestirring,andtrynottointroduceairintothesuspension.

Mitochondriakeepbestwhenconcentrated,tominimizeexposuretooxygen.Theyremaindormantuntildilutedintoanoxygen-richrespirationmedium.ThepasteshouldbetransferredtoanEppendorftube,andairbubblesavoidedbycarefulpipettingwithamicropipettorsetto,say,100µlorso.Thesuspensionsareveryviscous.Caremustbetakentoallowpressuretoequalizeafterdrawingupsuspension,otherwiseitmayshootupintothepipettor.Useofapasteurpipetatthispointresultsinlossofmuchofthepellet,sincethematerialreadilystickstoglass.Oncetransferredtoaneppendorftubethepreparation(whichmustbestoredonice)isreadyforuse.