Pleasenote:lately(2005-present),wenormallyculturePtK1cellsinF-12media,buttheyalsogrowinothertypesofmediaasdescribedbelow.

MixingF-12(HAM)Media*

(*Sigmastoppedmakingthepowderedmediaweusedinthepast-Sigma#N-4388;sowenowusepowderedmediafromGibco#21700-075orliquidmediafromGibco#11765-054.NeitherofthesehaveHepesbuffering)

1)StartbyrinsingagraduatedcylinderthreetimesddH₂O.2)Thenfillthecylinderto600-mlwithddH₂O.3)AddonepacketofF-12(HAMS)(Gibco#21700-075).4)Stirthisuntilallofthepowderisdissolved.(Note:Donotheattoquickentheprocess.)5)Onceallofthemediaisdissolved,add1.176-gNaHCO3(SodiumBicarbonate)andstiruntildissolved.6)NextpHthesolutionto7.15with1NNaOH.7)Bringthevolumeupto890-mlwithddH₂O.8)Splitthesolutionintotwoportions,eachbeing445-ml.9)Sterilefiltereachoneseperately.(Thisgivesyoutwoincompletebatchesthatcanbestoreduntilneeded).CheckthepH,itshouldbeat7.2-adjustaccordingly.10)Whenthemediaisneeded,add50-mlofFBS(FetalBovineSerum)and5-mlofantibioticstooneoftheportions.Thiscompletesthemediaforuse.

MEMforGFPhistonePtK1s1)rinsecylinderwellwithddH₂O2)add600mlsddH₂O3)addonebottleMEM(Sigma#M-0643)4)add10uMHepes5)pHto7.2with1NNaOH6)bringupto900mlswithddH₂O-testpH7)add5mLsofNEAA(non-essentialaminoacids,keepthissterile)8)450mlsmediapersterilefiltercontainer9)re-pHsmallsamplepouredintoFalcontubetoassurefilteringdidnotchangepHofmedia(thissometimeshappens)10)addfungizone,antibiotics(penandstrep)andFBStoonelabelcompleteMEM11)justaddmediatootherandlabelincompleteMEM12)addG-418to60ug/mlforGFPhistoneselection.(Youmaywanttoaddthisindividuallytosmallvolumesofmediaforusedependingontheamountofmediayou"llgothroughperunittime.TheG-418isn"tverystabletorepeatedheating/cooling.)

MEMforPtK1s1)rinsecylinderwellwithddH₂O2)add600mlsddH₂O3)addonebottleMEM(Sigma#M-0643)4)add0.85gSodiumbicarbonate5)add5mlsSodiumPyruvate(keepthissterile)6)pHto7.2with1NNaOH7)bringupto900mlswithddH₂O-testpH8)450mlsmediapersterilefiltercontainer9)re-pHsmallsamplepouredintoFalcontubetoassurefilteringdidnotchangepHofmedia(thissometimeshappens)10)addfungizone,antibiotics(penandstrep)andFBStoonelabelcompleteMEM11)justaddmediatootherandlabelincompleteMEM

DMEMforPtK1s1)rinsecylinderwellwithddH₂O2)add600mlsddH₂O3)add1bottleDMEM4)add3.7gSodiumbicarbonate5)pHto7.2with1NHCl6)bringupto900mlswithddH₂O-testpH7)450mlsmediapersterilefiltercontainer8)re-pHsmallsamplepouredintoFalcontubetoassurefilteringdidnotchangepHofmedia(thissometimeshappens)9)addfungizone,antibiotics(penandstrep)andFBStoonelabelcompleteDMEM10)justaddmediatootherandlabelincompleteDMEML-15forPtK1s1)rinsecylinderwellwithddH₂O2)add600mlsddH₂O3)add1bottleL-15(Sigma#L-4386)4)add7mMHepes(1.822gfor1L)5)pHto7.2withNaOH6)bringupto900mlswithddH₂O-testpH7)450mlsmediapersterilefiltercontainer8re-pHsmallsamplepouredintoFalcontubetoassurefilteringdidnotchangepHofmedia(thissometimeshappens)9)addfungizone,antibiotics(penandstrep)andFBStoonelabelcompleteL-1510)justaddmediatootherandlabelincompleteL-15

PhenolRed-FreeL-15withHighGlucoseforfilmingPtK1s1)BuyGibcoLeibovitz"sL-15mediumwithL-glutamine,withoutphenolred,cat.#21083-0272)Add4.5g/L(2.25g/bottle)glucose(Sigma#G-6152).3)Addfungizone,antibiotics(penandstrep)andFBS.

SplittingCells

1. Setupyournewflasksandcoverslipspriortodoinganythingtothecellsreadytobesplit.
2. Usingthevacuumtube,removetheoldmedia.
3. Rinsethecellswith6-7mlofSterilePBSandallowtositforafewseconds.
4. Usingthevacuum,removethePBS.
5. Put1.0mloftrypsinintheflask,moveflaskaroundandhititonthebottemagainstthetable(hood)todislodgefloatingcells.
6. Usingthevacuum,removethetrypsin.
7. Put1.0mloftrypsinintheflask,moveflaskaroundandhititonthebottemagainstthetable(hood)todislodgefloatingcells.
8. Usingthevacuum,removethetrypsin.
9. Placebackintheincubatorandallowtosituntilcellsarefloating.Timewillvarydependingoncellline~about4minutes.
10. Duringthisperiod,readythenewflasksanddishes.Put7-mlmediainthenewflasksand3mlinthedishes.Leavethecapsloose.
11. Whenyouremoveyourcellsfromtheincubator,bangtheflaskonacounteroragainstyourhandtoseparatethecellsfromoneanother.Observethemtomakesuretheyarefreefloating.
12. Toremovethecells,draw5mloffreshmediaandwashitoverthebottomoftheflask.Drawupthesame5-mlandrepeattwoorthreetimes.Nextkeepingthetipofthepipetinthefluid,drawupmostofthemediaandthenflushitbackoutintothemediaitself.Thendrawitallupandwashthebottomonefinaltime.
13. Drawupalloftheliquidandputthedesiredamountinyournewcontainers.Forourlab,usingPtK₁cells,thestandarddistributionisina1to3or4ratioforanewflaskand5-9dropsforacoverslipdish.Againthisratiowillvarywithyourcellline.

Useofciprofloxacin-treatedmedia:

Ciprofloxacinisaneffectiveantimycoticagentthatcanbepurchasedfrommosthospitalpharmaceuticalstockrooms.ItisoftenfoundunderthenameCiproI.V.,producedbyBayerCorporation.Itdoesaquiteanumberonfungiandmycobacteria,leavingtissueculturecellsfreeoftheirawfulinfluence.Twohundred-milligramCiproisdilutedfrom10mg/mLstockto10uL/mL(i.e.1:1000)incompleteF-12medium(includingbothFBSandtheusualpen/strep/ampmixture).Beforeintroductionofdrug-treatedmediumtoaflaskorpetridish,oldmediashouldbesuckedoff,andcellsshouldberinsedwithsterilePBS.Cellscanthenbefedorsplitasusual,usingtheciprofloxacinmedium.Acelllineshouldbesubjectedtothistreatmentforoneweek,startingfromthefirstdayofintroductionofdrug-treatedmediumbyfeedingorsplitting.Itisimportantthatflasksbelabeledastowhenthefirstdayofdrugtreatmentwas,sothatthetreatmentisnotcarriedonfortoolongaperiodoftime.Whentheprescribedperiodoftimehaselapsed,cellsshouldbeexaminedforthecontinuedpresenceofforeignorganisms;thisisdonemosteffectivelybystainingcellswithDAPI(aDNA-intercalatingdye),asbugswillappearaslittlespotsexternaltoyourcells"nuclei.Treatmentshouldberesumedforanother3-4daysifyoudoseebugsduringtheinitialcheck-up,anddiscontinuedimmediatelyifnoforeignorganismsaredetected.

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