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/EDTA4.3mMSodiumPhosphate,Dibasic(Na2HPO4)

pH7.2

2.7mMKCl

1.8mMPotassiumPhosphate,Monobasic(KH2PO4)

5mMEDTA

137mMNaCl

PBS4.3mMSodiumPhosphate,Dibasic(Na2HPO4)

pH7.2

2.7mMKCl

1.8mMPotassiumPhosphate,Monobasic(KH2PO4)

AlsoseeProtocol#2152

137mMNaCl

Ficoll-Hypaque1077Incubate15mlinfour50mltubes

Mustbeatroomtemperature

Equilibratetoroomtemperature

Dextran/PBS6%(w/v)DextranT500(Pharmacia)

PrepareinsterilePBS(Ca2+,Mg2+free)