• PBS(Phosphatebufferedsaline)pH7.4
• Saponin(Sigma)-make10mg/mlsaponininPBS.Addazide&storeinrefrigerator.
• Blood
• Antibody

Collectbloodwithalargegaugeneedleintoasyringe.Donotsubjectthesampletolargestressesbypullingtoohardonthesyringe.Ifyoudothisyouwillcertainlydamageerythrocytesandprobablyalsodamageleukocytes.

Youmaycollectthesampledirectlyintoanticoagulantinthesyringe.Ifyoucollectinasyringewithoutanticoagulant,carefullyintroducethebloodintoatubecontaininganticoagulant.Donotallowthesampletofrothineithercase.

HOWEVERYOUCOLLECTTHEBLOOD,ENSURETHATITISCOMPLETELYMIXEDWITHANTICOAGULANT.MIXBYINVERSIONATLEAST10TIMES!

Ifyouareconductingastudyofpatientgroupsitisdesirabletodrawsamplesfrommatchedcontrolsubjectsatthesametime.Trytomakesurethatallbloodisdrawnatasimilartimeofday;ifnotpossIBLe,recordthetimeofdayitwasdrawnanduseanappropriatecontrolsubject.

Ifyouareinterestedin"resting"levelsofantigens,immediatelyputthetubeofbloodintoice.KEEPCOLDTHROUGHOUT.

DividethebloodintotheappropriatealiquotsandaddlabeledmAbtoeachaliquotasdesired.MakesurethatthefinalmAbconcentrationisatsaturation-youmayneedtodeterminethisbytitration.Doblockedsamplesasnecessary.

Incubate30minutesonice.

Notethatthisisfordirectlylabeledantibody.Ifyouneedtouseanindirecttechnique,youwillneedtodowashesandincubatewithsecondaryreagentatthispoint.Prepareastocksolutionofsaponin,10mg/mlinPBS.Add0.1%sodiumazideandstoreintherefrigerator.

Beforethemeasurementphaseoftheexperimentstarts,prepareasufficientquantityoftubeseachcontaining0.05mlsaponinsolution.Placetheseoniceandallowtocool.

Add0.025mlbloodtoa0.05mlaliquotofsaponinandmixwiththePipettetip.Wait5secondsthenadd0.45mlPBS.Periodicallyagitateforashortperiod,upto30seconds,monitoringsampleclarityvisually.Itiseasytotellwhentheerythrocyteslyse,becausethesampleclearsrapidly.Assoonaslysishasoccurred,returnthetubetoice.Allowafurther10secondstocompletethelysisandreadontheflowcytometer.Usecharacteristicforwardversusorthogonallightscatteringtosetagateforthetypeofcellsinwhichyouareinterested.Acquireatleast5000eventsandstorependingoff-lineanalysis.

Youcanaddupto0.05mlbloodtoeachsaponinaliquotwithgoodresults.Donotaddmore,otherwisetheerythrocyteswillnotlyseproperly.

Itseemsthathumanleukocytesarequiteresistanttosaponinlysis,andthesampleswillbestableoniceforabout3-5minutes.Otherspeciesdifferinthisrespectandyoumayneedtomodifythelysisprotocoltosuit.Ihavehadgoodresultswithhuman,rabbit,rat,baboonandpig(butnothamster).ForoptimumleukocytestABIlityitmaybenecessarytokeepthesamplecoldwhileitisrunningonthecytometer(useanicebath).

Theconcentrationofsaponinusedheredoesnotappeartoremoveantigensfromthecellmembrane,howevertestthiswithpurifiedcellsifyouaresUSPicious.