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Human Peripheral Blood Mononuclear Cell Preparation

来自 : 蚂蚁淘

Thisprotocoldescribesaprocedureforisolatinghumanperipheralbloodmononuclearcells(lymphocytesandmonocytes)fromaBuffyCoat(obtainedfromabloodbank).Platelets,redbloodcells,andneutrophilsarelargelydepletedfromthefinalcellsample.Theisolatedcellscanbeusedinchemotaxisassays

Procedure

1.Pourbuffycoat(seeHint#1)fromthebagintoa125mlconicalpolypropylenecentrifugetubeanddiluteto90mlwithsterileendotoxin-freePBS.2.Add15mlofsterileDextran/PBSandmixbyinvertingthetubefourtofivetimes.Ifanybubblesappear,theyshouldberemovedwitha1mlplasticPipette(seeHint#2).3.Leavethemixtureundisturbedatroomtemperaturefor20mintoallowtheerythrocytestosediment.4.TransferthesupernatantwithaplasticpipettetoafreshtubeandaddanequalvolumeofPBS/EDTA.5.Centrifugeat1,000Xgfor10minatroomtemperaturetopellettheleukocytes,leavingtheplateletsinsUSPension.6.Pourthesupernatantintoacontainerofbleach(seeHint#1),resuspendthecellpelletin1to2mlofPBS,andtransferthelattertoafreshtube(toavoidcontaminatingthesolutionwiththeplateletsthatareadheringtothesidesofthetube).7.Bringthecellsuspensiontoafinalvolumeof50mlandgentlyoverlay12.5mlontotheFicoll-Hypaqueinfour50mlconicaltubes(15mlFicoll-Hypaquepertube).8.Centrifugeat800Xgfor20minat12°CwiththeBRAKEOFF.9.ThemononuclearleukocytesshouldappearasacloudyringatthePBS/Ficollinterface.Harvestthemfromtheinterfacewitha2or5mlpipette.10.Transferthecellsfromeachinterfacetoafresh50mltubeandfillwithPBS.11.Centrifugethesuspensionat800Xgforaminimumof10mintopelletthecells.Aspiratethesupernatantandresuspendthecellpelletsinappropriatebuffer(seeHint#3).Solutions

PBS/EDTA		4.3mMSodiumPhosphate,Dibasic(Na₂HPO₄)pH7.22.7mMKCl1.8mMPotassiumPhosphate,Monobasic(KH₂PO₄)5mMEDTA137mMNaCl

PBS		4.3mMSodiumPhosphate,Dibasic(Na₂HPO₄)pH7.22.7mMKCl1.8mMPotassiumPhosphate,Monobasic(KH₂PO₄)AlsoseeProtocol#2152137mMNaCl

Ficoll-Hypaque1077		Incubate15mlinfour50mltubesMustbeatroomtemperatureEquilibratetoroomtemperature

Dextran/PBS		6%(w/v)DextranT500(Pharmacia)PrepareinsterilePBS(Ca²⁺,Mg²⁺free)

BioReagentsandChemicals

EDTAPotassiumPhosphate,MonobasicFicoll-Hypaque1077DextranT500SodiumPhosphate,DibasicPotassiumChlorideSodiumChloride

ProtocolHints

1.CAUTION!Thissubstanceisabiohazard.Consultthisagent"sMSDSandinstitutionalpoliciesforproperhandlinginstructionsforhumanblood.2.Thebubblestrapredbloodcells.3.Todepletemonocytes,culturethemononuclearcellmixtureinseveral10cmdishesfor30minutesto1hour.Monocytesadheretotheplasticdishandthenonadherentlymphocytescanbetransferredtoanewtube.