嗜酸性粒细胞的提纯---医学期刊频道--首席医学网

2004-06-08;2004-09-23(1974-),,,,,Isolationandpurificationofeosinophilicgranulocytes,,(,300162):;;;100825041(2005)0120062205R32912B015%5%,015109/L(),10%,,,,1,1109011095g/ml;1108011085g/ml;1105011066g/ml;1105211077g/ml;11091111g/ml;1103011060g/ml1,Fi2coll2HypaqueMetrizamidePercoll111Ficoll()2Hypaque()(polysucrose,Ficoll),,,40000011111:(10/ml),5%FCS(fetalcalfserum,)/Hanks,(11077),1000g,20min,,515min,400g,7minHanks12,2ml5%/Hanks30min,371h2000rpm15min2,3,Hanks2,1000g10min,11112:HanksFicoll(400)Hypaque15%Ficoll20%Hypaque11113:1108110911105g/mlFicoll2Hypaque2ml,,20,1200g45min,Hanks3Wright()2Giemsa(),Trypan2blue(),85%95%,40%70%2%,2112MetrizamideMetrizamide(,,)11211:1111111212:30%MetrizamideTy2rodesgel/NDase,l11611179g/ml,540mOsm,Tyrodesgel/DNase,18%25%Metrizamide,11101113g/ml11213:24%23%22%20%18%Metrizamide,2ml,201200g45min,MEM/FCS/DNase90%,50%26ActaAcademiaeMedicinaeCPAPF141Vol114No1120051Jan.2005,,540mOsmMetrizarmdeMetrizamide,;2%,4113PercollPercollpharmacia,(PVP),20mOsm/(kgH2O),,113g/ml,(2001000g)Percoll,,Percoll,,,,,11311:1111111312[5]:Percoll,PBSPercollPBSd1x+d2(y2x)=d3y11100,11090,11085,11080g/mlPercoll,4(d1Percoll,d2PBS,d3,xPercoll,y)11313:12cm15cm,11100,11090,11085,11080g/mlPercoll,,10001600g20min,Hanks,Wright2Giemsa,Typan2blue11090g/ml90%6,4%,38%56%7Percoll,Ficoll2HypaqueNH4ClPercoll,(pH710)30min,8Percoll1108611100,Eos86%,38%56%,11086EosPercoll11086,Eos,Eos,Eos2[9],,,11082g/ml11094g/mlPercoll,10CD16,,CD16IMB(ImmunomagneticMicrobeads,IMB),IMB,MACS(MagneticActivatedCellSorter,),,,;,11(cocktailmethod),,,,,1,211(),SPA(A,SPA,Fc),4,,,5()(1mol/LpH=810),1h,,PBS,CD16,41h,PBS,CD16,4212,,PBS,20ml11083g/mlPercoll(12,13),1000g30min,5155mmol/LNH4Cl,015min,PBS(2%FCS)2,47min(400g),10l,90lPBS(2%FCS),(=201),41h36141Vol114No1120051Jan.2005ActaAcademiaeMedicinaeCPAPFPBS4ml(2%FCS),MACS,50ml,PBS32ml(2%FCS),,400g7min,,,:(1),Percoll4%,Metriza2mide2%;(2)CD16+,MACS9915%011%,80%;(3),,,14,153(FlowCytometry,FCM)(),,,,,,,,,,,4,/s,,(10ms),,,16,,(45)106/h,(90%99%),,95%17,;,,,,,FCM,,18,,CD16FITC,194411,,,,,,30%20,,,30s,(pH714,155mmol/LNH4CL,10mmol/LKHCO3,011mmol/LNa2EDTA),4,21412percoll,,;,,,,;,,,,,,;,pHpH,pH710percoll,11082g/ml,1108211100g/ml,pH714percoll,11095g/ml,46ActaAcademiaeMedicinaeCPAPF141Vol114No1120051Jan.20051109511105g/ml413,,,,,,,10%,,,,,,,414,;,,165Ficoll2HypaqueMetrizamide,,2%,Metrizamide,,Percoll,FCMMACS,,,Kuni2hikoKpercoll,90%6,95%98%,98%5Percoll4%1108211100PercollEos96%,Eos380,,CD4CD8CD3422,,,,,FCMMACS,,,,,11M.:,2002.61-68.2VadasMA,DavidJR,BulterwcrthA,etal.AnewmethodofpuriticationofhumanneutrophilsandeosinophilsJ.JImmunol,1979,122(4):1228-1235.3,,,.J.,1999,21(10):763-764.4HanselTT,PoundJD,ThompsonRA.IsolationofeosinophilsfromhumanbloodJ.JImmunolMethods.1990,127(2):153-64.5.J.,2001,18(6):917.6KunihikoK,SatoruN,KenichiroH,etal.AugmentationofapoptosisinbronchialexudedrateosinophilsbycyclosporinAC.BiochemicalandBiophysicalResearchCommunication,1996,222:71-77.7LavioletteM,BosseM,LavigneS,etal.ComparisonoftwomodifiedtechniquesforpurifyingbloodeosinophilsJ.JImmunolMethods,1993,165(2):253-261.8YazdanbakhshM,EckmannCM,RoosD.Characterizationoftheinteractionofhumaneosinophilsandneutrophilswithopsonizedpar2ticlesJ.JImmunol,1985,135(2):1378-84.9,,.J.,2000,16(8):423-427.10Zeck2KappG,KroegelC,RiedeUN,etal.Mechanismsofhu2maneosinophilactivationbycomplementproteinC5aandplatelet2activatingfactor:similarfunctionalresponsesareaccompaniedbydifferentmorphologicalterationsJ.Allergy,1995,50(1):34-47.11ShinagawaK,AndersonGP.RapidisolationofhomogeneousmurinebronchoalveolarlavagefluideosinophilsbydifferentiallectinaffinityinteractionandnegativeselectionJ.JImmunolMeth2ods,2000,237(1-2):65-72.12SzucsS,VargaC,EmberI,etal.Theseperationofthegranu2locytesfromdifferentratstrainsJ.JImmunolMethods,1994,167:245.13ScribaA,LucianoL,SteinigerB.High2yieldpuriticationofratmonocytesbycombineddensitygradeientandimmunomagneticsep2arationJ.JImmunolMethods,1996,189:203.(71)56141Vol114No1120051Jan.2005ActaAcademiaeMedicinaeCPAPF1,,,.34J.,1993,9(4):202-204.2.M.:,2002.1370-1374.3IvaturyRR,NallathambiMN,RohmM,etal.Penetratingcar2diactrauma:quantifyingtheseverityofanatomicandphysiologicinjuryJ.AnnSurg,1987,205:61-66.4.M.:,1996.741-746.5,,,.J.,2004,20(5):270-272.6,,,.52J.,1998,14(3):190-191.7,,.M.:,1998.754-758.8,,.M.:,2001.498.9,,,.56J.,2000,38(5):358-359.(:)(65)14StanciuLA,ShuteJ,HolgateST,etal.ProductionofIL28andIL24bypositivelyandnegativelyselectedCD4+andCD8+humanTcellsfollowingafour2stepcellseparationmethodinclud2ingmagneticcellsorting(MACS)J.JImmunolMethods,1996,189:107.15RubbiCP,PatelD,RickwoodD.EvidenceofsurfaceantigandetachmentduringincubationofcellswithimmunomagneticbeadsJ.JImmunolMethods,1993,166:233.16,.M.:,1998.178-179.17,.M.:,2000.158.18.M.:,2002.78.19SandstromK,HakanssonL,LukiniusA,etal.AmethodtostudyapoptosisineosinophilsbyflowcytometryJ.JImmunolMethods,2000,240(1-2):55-68.20TaiPC,SpryCJ.PurificationofnormalhumaneosinophilsusingthedifferentbindingcapacitiesofbloodleucocytesofcomplexedrabbitIgGJ.ClinExpImmunol,1977,28(2):256-263.21FukudaT,DunnetteSL,ReedC