CMFDA Labeling of Platelet

OUTLINECMFDA(5-chloromethylfluoresceindiacetate)isalipophilictracerthathasanenormousadvantageoverordinarytracers(e.g.FITC)becauseitcanbeintroducedintolive(!)cells.Onceinsidethecellthisdyeundergosomecovalentchangesrestrictingfromitspassgaetoextracellularmilieu.PROTOCOL

1. Collectbloodandpurifyplatelets.
2. ResUSPendinthecertainknownvolumeofBSGCandcountplatelets.Separate10x10^6platelets(negativecontrol).Centrifugeat3000rpmat4ѓC,for7min.StorethemresuspendedinIscove"sModifiedDulbecco"sMedium(IMDM),at4ѓCbeforetheFACSpreparationstarts.
3. Resuspend3.2x10^8plateletsin5mlofBSGCtogetherwith50µgofCMFDAin15mlFalconTube.Storefor60min.,atRT,atdarkwithslightrotation.
4. Centrifugeat3000rpmat4ѓC,for7min.
5. Resuspendin5mlofBSGC.
6. Centrifugeat3000rpm,at4ѓC,for7min.
7. Lettostayfor60min.,atRT,atdark(eliminationofCMFDAaccess)
8. Centrifugeat3000rpm,at4ѓC,for7min.
9. ResuspendinthecertainknownvolumeofBSGCandcountplatelets.Separate10x10^6platelets(qualitycontroloflabeling).Centrifugeat3000rpmat4ѓC,for7min.StorethemresuspendedinIMDM,at4ѓCbeforetheFACSpreparationstarts.
10. Themainpartofthelabeledplateletsshouldbecentrifuged,resuspendedintherespectivesolution(NaCl0.9%forinvivouse,IMDM,HCForPBSforinvitrouse),counted,andutilizedinsubsequentinvitroorinvivoexperiments.

1. BSGC
2. IMDM(Iscove"sModifiedDulbecco"sMediumwith25mMHepes/withL-Glutamine)BioWhittakerEuropeCat.:BE12-722F

1. WeuseCMFDAspecialpackfromProbes.
2. [readmore]aboutCMFDAdye.

1. Baker,G.R.andLevin,J.(1998).TransientthrombocytopeniaproducedbyadmiNISTrationofmacrophagecolony-stimulatingfactor:investigationsofthemechanism.Blood91(1):89-99.
2. Levin,J.,Peng,J.P.,Baker,G.R.,Villeval,J.L.,Lecine,P.,Burstein,S.A.andShivdasani,R.A.(1999).PathophysiologyofthrombocytopeniaandanemiainmicelackingtranscriptionfactorNF-E2.Blood94(9):3037-47.