植物生物反应器在制药中的应用
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YeastCellCyclebyFlowCytometry
SusanForsburg"smethodforSchizosaccharomycespombe
- Coldabsoluteethanol.
- 0.5MNacitratestock(filtered),50mMdilutedstock.
- 10mg/mlRNaseA(Boil10mins,cool,filterandstoreat-20°C).
- 4mg/mlPropidiumiodide(PI)(filterandstoreindarkat-20°C).OR
- SYTOXGreen(MolecularprobescatalogS-7020;5mMstockinDMSOandstoredindarkat-20°C)
- Spindown107cellsfromanexponentiallygrowingculture-2000rpmfor5mins.Pouroffsupernatant.
- Vortextubewhileadding1.0mlcold70%EtOH.
- Storeat4°C(cellskeep~indefinitely).
- Whenyouwanttoprocessthecells,take0.3ml(thiswillbe2-3x106cells,assumingalittlelossinthewashing)andaddto3ml50mMNacitrateina5mlFalcontube.Mixandspin2000rpmfor5mins.
- DiscardsupernatantandresUSPendpelletin0.5ml50mMNacitratecontaining0.1mg/mlRNaseA.Leavein5mlFalcontubeandputin37°Croomfor2h.
- Forstaining:
- PropidiumIodideAdd0.5ml50mMNacitratecontaining8µg/mlPI,sothatfinalconcentrationinthesampleis4µg/ml.Therecanbenon-specificstainingofyeast(pombe)endsathigherconcentrationsifcellsarestarved,orspores.Cellscanbeprocessedimmediatelyorconvenientlystoredovernightat4°Cinthedarkbeforeprocessingthenextday.Ifnecessarycellscanbestoredatthisstageforamaximumofaweek(4°Cinthedark).Checkthemunderthefluoresencemicroscope(redchannel)toverifystaining.
- SytoxGreenAlternatively,add0.5ml50mMNacitratecontaining2µMSytoxGreen,sothatfinalconcentrationinthesampleis1µM.
- (Optional)Justbeforeprocessingthecells,sonicatefor45sagainleavingcellsinthe5mlFalcontubes.SonicationpreventsdoubletsofcellswhichgivespuriouspeaksandisparticularlyusefulifyourcellshavevaryingDNAcontentsandwillcleanupsporesorweemutants.
- ApproximatesettingsontheFACScanforPropidiumIodide
- DetectorFSCE00Gain:3
- DetectorFL2-AVoltage:890Gain:2
- ApproximatesettingsontheFACScanforSytoxGreen
- DetectorFSCE00Gain:2
- DetectorFL1-AVoltage:400Gain:4
Pointstobearinmind
- Youcanfixmorethan107cells,butdon"tprocessmanymorethan5x106fixedcells.Usingtoomanycellscanleadtoincompletestainingandartefacts.
- Youcanmakecontrolsrepresenting1,2and4CDNAcontents.Usenitrogenstarvedhaploidcells,exponentiallygrowinghaploidsandexponentiallygrowingdiploidcellsrespectively.Youcanfixlargenumbersofcellsandusethemovermanymonths.It"shelpfultoincludeacontrolsampleineachseriesofsamplesthatyouprocess.
- Ethanolfixedcellscanbesentinthepostatroomtemperaturewithoutcomingtoanyharm.StainedcellscanbeFedEx"dwithoutcomingtoanyharm.
- Ifyouaredealingwithparticularlyfragilecells(e.g.veryelongatedcells)theremaybeaproblemwithlysiswhencellsarewashedinwaterbeforefixation.Thiscanbeavoidedbywashingwith1Msorbitol.Youcanevenfixcellsin70%ethanol,30%1MSorbitol.Ifyouhaveproblemswithlysisevenintheculturemedium,then1.2Msorbitolcanbeincludedhereaswell.WashoutthesorbitolbeforeflowcytometricanalysisbecauseitdestABIlizesthesamplestreamresultinginhighCVs.
- Learnhowtousethe"LiveGate"option.Thisallowsyoutoreducethebackgroundinyoursamples(whichmaybecausedbyanythingfromparticlesofmediumtobacteriaorothercontaminants)andwillimproveyourdata.Italsogivesyoutheoptionoffocusingonaparticularsubpopulationthatyoumaybeinterestedin.
Generalreference(PImethod):SazerandSherwood(1990)J.CellSci97:509-516
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