BloodHarvest

·Anesthetizemicewith50mlofXylene-Ketamine(2:1)coctail

·Pinanimaltoaboard(usedistalextremities)supine

·Use70%alcoholtomoistentheaBDomenandlowerthorax

·Makeanmidlineincisionusingforcepsandscissorsfrommidabdomentoxyphoidprocess

·Dissecttotheleftsideoftheanimalandopentheperitoneum,exposingtheliveranddiaphragm

·Lifttheanteriorchestwallwithaforcep,theheartshouldbeapparentthoughthetranslucentdiaphragm

·Filla1ccsyringewith300mlofheparin

·Holdingthesyringecomfortably,withoneconcertmotioninserttheneedleintotheheartthroughthediaphragmanddrawblood

·Shouldthefirstattemptnotsucceedandpneumothoraxdevelops,immediatelyopenthechestcavitythroughamidlineincision(sternotomy)anddrawblooddirectlyfromtheheart

Labelinglymphocytesandseparatinglymphocytesfromthewholeblood

·Add250mlofDMEMwith10%FetalGoatSerum(FGS)to250mlofwholebloodtoblocknonspecificbinding

·Incubateat4°Cfor10minutes

·Add1mgofappropriateantibodiestothespecimen(usuallyaboutamillioncells)

·Incubateat4°Cfor30minutes

·Add2mlofcoldlyticbuffersolutiontoeachspecimenfor2minutes–gentlyshakethem

·After2minutesfillthefacstubeswithPBS

·Spinat1000rpm(450g)for10minutesat4°C

·Aspiratesupernatanttopalletusingvacuumsetup

·Add2mloflyticbuffersolutionforthesecondtimefortheresidualRBCsandfilltubeswithPBS.Spinagainfor10minutesat1000rpmat4°C

·Aspiratesupernatantwithvacuumsetuptopallet

·Add?mlofPBStothepalletsandgentlymixthem

·Specimensarereadytobereadbythefacsmachine.Theycanbestoredat4°Cforatleast2days.