Isolation of PBMCs from patient blood and buffy coats

1|Transferheparinizedvenousbloodofpatienttoplastic50-mltubesanddilutewithanequalvolumeofPHbuffer.

Whensodiumcitratehasbeenusedasanticoagulant,addanequalvolumeofPTbuffer.WhenPBMCsareisolatedfromabuffycoat,transferthebuffycoattoa250-mlflaskandaddPTbuffertoafinalvolumeof150ml.

2|Gentlyload25mlofdilutedbloodontopof12.5mlofaFicoll-Isopaquesolutionwithadensityof1.077gml
1ina50-mltube.

3|Centrifugeat760gfor20minatroomtemperature(21±51C).Slowaccelerationanddonotusebrake.

4|CollectthecellbandontopoftheFicolllayerwithaPasteurPipetteandtransferittoanew50-mltube(maximumof2cellbandspertube)andaddPHorPTbuffertillafinalvolumeof50ml.Centrifugeat425gfor15minatroomtemperature.

5|Washtheharvestedcellstwiceinatotalvolumeof50mlofPHorPTbufferandcentrifugeat425gfor10minatroomtemperature.ItisimportanttocompletelyresUSPendthecellpelletforeachwashtoensurethrombocyteremoval(seealsobelow).

6|ResuspendthecellsinIFmediumandcountthecellsusinganautomatedcellcounter.

CAUTIONWorkingwithprimarycellsfromblooddonorsandHIV-1infectedpatientsmayresultinthefrequentintroductionofmycoplasma.Precautionsshouldbetakentopreventinfectionofothercellsorcelllines.Itisrecommendedtoaddciprofloxacintothecultures,toinhibitreplicationofmycoplasma.

CRITICALSTEPThepresenceofthrombocytesinthePBMCscultureswillhaveanegativeeffectonisolationofHIV-1.Whenthesupernatantisnotclearafterthesecondwash(Step5),largeamountsofthrombocytesarestillpresentinthePBMCs,andadditionalwashsteps(repeatStep5)shouldbeaddedtoremovethethrombocytes.

PAUSEPOINTPBMCscannowbeeithercryopreservedinIMDMcontaining10%(vol/vol)FCSand10%(vol/vol)DMSOordirectlyused.CryopreservedPBMCscanbestoredinliquidnitrogenformorethan10years.

Determinationofa32bpdeletionintheCCR5geneofPBMCsdonors

7|IsolategenomicDNAfrom1106PBMCsor200mlofbloodusingtheQiagenbloodkitorequivalentalternative,accordingtothemanufacturer’sinstructions.

8|PreparethePCRmixasfollowsusingprimersCCR5-sense(position427to450inCCR5(NM_001105536):5￠-GATAGGTACCTGGCTGTCGTCCAT-3￠)andCCR5-antisense(position665to644inCCR5(NM_001105536):5￠-ACCAGCCCCAAGATGACTATCT-3￠)

flankingthedescribed32-ntdeletionintheCCR5gene.

ReagentAmount(ll)

PCRreactionbuffer(10)5

dNTP(5mM)2

MgCl(50mM)2.6

PrimerCCR5-sense(100ngml
1)1

PrimerCCR5-antisense(100ngml
1)1

TaqDNApolymerase0.2

H2O33.2

Totalvolume45

9|Add5ml(10–100ng)ofgenomicDNAtothereaction.

10|AmplifythePCRproductsunderthefollowingconditionsinaThermocycler:

StepTemperature(1C)TimeNumberofcycles

1955min1

2955s35

5510s

721min

3722min1

44N1

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