erythrophagocytosis assay
来自 : 蚂蚁淘
OUTLINETheerythrophagocytosisassayisperformedtocomparethephagocyticratewithandwithoutanti-eythrocyteantibodyineithermacrophagesfromcontrolanimalsorvirus-infectedcounterparts.
PROTOCOL[erythrophagocytosis]- prepareperitonealmacrophages.
- countperitonealcellsanddistributeon6-wellsGrenercellcultureplate(2-6x10^6peritonealcells/ml),placeinThermostateat37ѓC,6-7%pCO2for3hours.
- aspiratethesupernatant(SN),add2mlofIMDM-AJ(3-5%)-FCS/FBS(5-10%)andaspiratetheSN,repeat1moretime(washingprocedure).
- youmayruntheerythrophagocytosisrightnowor,alternatively,changetheculturemediumandplacemacrophagesinthermostateat37ѓC,6-7%pCO2,ON(overnigh).Nextdaywashonceandincubatewithpreparederythrocytes(RBC)for1-3hoursinthermostateat37ѓC,6-7%pCO2.
- wash3moretimes,add1mlofsterilePBS(ice-cold,tostopphagocytosis).
- countmacrophageaspositiveif5andmoreRBCshavebeenphagocytosed(oninvertmicroscope).Estimatetherateofphagocytosisasaproportionofpositivecellstooverallcalculatedcells,%.
[preparationoferythrocytes]- collectbloodonheparine.
- centrifugeat1000rpm,10ѓC,7min.
- resUSPend2mlofRBCsin5mlofPBS(sterile,ice-cold),placeat4ѓC,ON.
- nextdaywash2timeswithsterilePBS(centrifugation-resuspensioninPBS).
- resuspend500µloftheRBCsin10mlofPBS-BSA(1%),add50µlofanti-RBCantibody,incubatefor1houratRT,applyslightrotation.
- wash2timeswithPBS(centrifugation-resuspensioninPBS).
- centrifugeat1000rpm,10ѓC,7min.
- take20µlofRBCpelletandaddtomacrophages.
SOLUTIONS- IMDM[(Iscove"sModifiedDulbecco"sMediumwith25mMHepes/withL-Glutamine)BioWhittakerEuropeCat.:BE12-722F]-AJ(3-5%)-FCS/FBS(5-10%)
- PBS
- PBS-BSA1%
- NH4Cl,160mM
ADDITIONALINFO
- IMDMsupplementedwithAJ(3-5%),FCS/FBS(5-10%)andheparine(100U/ml)maybeusedwhenpurifyingmacrophages.
- anadditionalstepofosmaticshock(with1mlofNH4Cl,forlessthan3min)maybeaddedrightafterthefirstcentrifugationatthestepofmacrophagepreparationwithaconsequentneutralizationstepwithIMDM-AJ(3-5%)-FCS/FBS(5-10%)
- thehypotheticalratioofperitonealmacrophages/peritonealcellsis1/3.
- distinguishphagocytosedRBCsandoverposedones.
SCHEME
REFERENCESHunter,S.,Indik,Z.K.,Kim,M.K.,Cauley,M.D.,Park,J.G.andSchreiber,A.D.(1998).InhibitionofFcgammareceptor-mediatedphagocytosisbyanonphagocyticFcgammareceptor.
Blood91(5):1762-8.
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