DescriptionThisprotocolisusedinourlabtoreducethecostsofthecellsortingwithMACSreagents.ThecellsUSPensionobtainedafterthisprotocolcontains40-70%monocytes.ThiscellsuspensionisthanusedforpositiveornegativeMACSseparation.ProcedureBuffycoatstobeusedforthisprotocolmustbefreshandproducedwithoutplateletdepletion.ThevolumeofbuffycoatthatweobtainfromRedCrossisusuallyabout75ml.Note:PBSshouldbewithoutCa2+andMg2+!1.Dilutebuffycoatto150mlwithPBS.2.Prepare2Ficoll(Biocoll)grADIentsinLeucoseptubes(15mlFicollpertube).3.Apply30mlofdilutedbloodtoeachgradient.4.Centrifuge30minat400xgwithoutbrakes.5.CollectthePBMCfractionandtransferitintoa50mltube.6.FillthetubewithPBS,centrifuge10minat300xg.7.Resuspendgoodin50mlPBS,centrifuge10minat200xg.8.Resuspendgoodin50mlPBS,centrifuge10minat300xg.9.PreparecontinuousPercollgradient(seebelow).10.ApplyallthecellsonthePercollgradient.11.Centrifuge30minat400xgwithoutbrakes.12.CollectenrichedPBMCsandtransferintoa50mltube.13.FillthetubewithPBS,centrifuge10minat300xg.14.Resuspendgoodin50mlPBS,centrifuge10minat200xg.15.Resuspendgoodin50mlPBS,centrifuge10minat200xg.16.Determinethecellnumber,takeanaliquotforFACSanalysisandproceedwithMACSseparation.RecipesContinuousPercollgradientpreparation:1.Mixin50mltube*:13.5mlPercoll(Amerhsam),15.0mlMEMSpinnermodification(Sigma),1.5ml10xEarlessaltssolution(Biochrom)2.Centrifuge10minat14000xgwithoutbreaksinafixedanglerotor.3.Carefullytakethegradientoutoftherotor.Thegradientisreadytousenow.SuppliesTips*Thetubeshouldbeabletoresistcentrifugationat14000xg.