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11:FlowCytometry:SamplePreparationandStorage2•Visualizethesampletocytometer•Singlecellsuspension•Fluorescentlabelling•Preventorminimizeartefactsduringsamplehandling•Optimizeandstandizesamplehandlingprecedures3Cellsuspensionpreparationfromsolidtissuesandculturedcells•Mechanicaldissociation•Enzymaticdissociation4•→→→→→→→→→→→→→→→→••4°4°CC••••80µm80µm5•:→→→→→→→→→→→→→→→→•:••:2.5mg/ml:2.5mg/ml(trypsin),0.5mg/ml(trypsin),0.5mg/ml(collagenase)(collagenase)II,II,20µg/mlDNA20µg/mlDNA(DNase)I(DNase)I••:37°C,:37°C,11,,••80µm80µm••6(I)•••:5mMEDTA,30min:5mMEDTA,30min••::••::,,,,•••:trypsin/EDTA(0.02-0.1%/5mM),1:trypsin/EDTA(0.02-0.1%/5mM),1––10min10min••::,,,,••::2705101520ICAM-1Expression(arbitraryunit)EDTATrypsin/EDTACorverWE,etal.Cytometry1995;19:267-272.80200400600800ECVCAM-1Expression(arbitraryunit)↓↓GräbnerR,etal.Cytometry2000;40:238-244.9(II)•••::••::,,••::10:•,DNA•,DNA•EDTA:,DNA•:,DNA110.00.51.01.52.02.5CitrateEDTAHeparinHirudin*§***Unstimulated0.1µMfMLPLiN,etal.EurJHaematol2000;65:57-65.CD11bLeukocyteCD11bMFI12Pre-samplehandlingstorage:4°C:4°C•:,,,•≤1h,4°C:•2h,4°C:•≤24h,4°C:•≤72h:DNA313(4°C)LiN,etal.EurJHaematol2000;65:57-65.0.00.51.01.52.00.00.51.01.52.00.00.51.01.52.0Time(hour)0.00.51.01.52.0LymphocytesMonocytesNeutrophilsLeukocytes0136122401361224fMLP0.1µMUnstimulatedLeukocyteCD11bMFI14CD69(4°C)HalldénG,etal.AnnAllergAsthmaImmunol1999;83:413-421.15•••::••,DNA,DNA•••:DNA:DNA,,16LiN,etal.EurJHaematol2000;65:57-65.48(4°C)0.00.51.01.52.0CD11bExpressionMFITime(hour)Unfixed0.5%formaldehydefixed013612244817(I)•••••::;;••::;;,,•••,,••::;;••::18(II)•••••::••::•••••::••::419LundahlJ,etal.JImmunolMethods1995;180:93-100.20ForsythKD&LevinskyRJ.JImmunolMethods1990;128:159-163.21(I)––••••••••••22(II)––••••••TritonX-100,NP-40,Tween20,Lysolecithin,saponin,andOGTritonX-100,NP-40,Tween20,Lysolecithin,saponin,andOG•23LundahlJ,etal.APMIS1991;99:136-146.Control0.1%Triton0.8%OG24(III)––1••IgG••IgG,•/(F/Pratio)525(IV)––2•15,000g•Fc••••••F(ab)orF(abF(ab)orF(ab’’))22•F(ab)F(ab’)226StewartCC&StewartSJ.MethodsCellBiol2001;63:217-251.FITC-CD315min10min+FITC-CD315min27(V)––•••phycoerytherin(PE)PE-Cy5PE-TexasRed(phycoerytherin(PE)PE-Cy5PE-TexasRed(EECCDD))allophycocyanin(APC)fluoresceinisothiocyanate(FITC)allophycocyanin(APC)fluoresceinisothiocyanate(FITC)•••PEPE-Cy5FITCPEPE-Cy5FITC••FITC28(VI)––•:••::??••::??•“”••••29(VII)––••(37°C)•300.02.55.07.510.037°C22°C15°C4°C++***++++++LiN,etal.EurJHaematol2000;65:57-65.CD11bUnstimulated0.1µMfMLPLeukocyteCD11bMFI631•/••••Paraformaldehyde:0.5-4%inPBSParaformaldehyde:0.5-4%inPBS••Formaldehyde:0.2-1%insalineorPBSFormaldehyde:0.2-1%insalineorPBS••Glutaraldehyde:Glutaraldehyde:••••Ethanol:Ethanol:••Methanol:Methanol:••Acetone:Acetone:•32024682.0%PF1.0%PF1.0%F0.5%F0.2%FUnfixedUnstimulated02468Time(loghour)0.1µMfMLPLiN,etal.EurJHaematol2000;65:57-65.:CD11b480136122448LeukocyteCD11bMFI33•ThereisNOTagoldenprotocolforflowcytometricsamplepreparationthatsuitseveryapplication.•OptimalproceduresofsamplepreparationshouldalwaysbesetupandevaluatedINDIVIDUALLY.