Capacity:for20isolationsOverview
TheExosomeIsolationKitCD9orCD63orCD81,humanfacilitatesthespecificisolationofintactexosomesorextracellularvesicles(EVs)fromcellculturesupernatant,plasma,urine,orascites.TheisolationisperformedbypositiveselectionusingMicroBeadsrecognizingthetetraspaninproteinsCD9orCD63orCD81.Theisolationprotocolisbasedontherenowned
MACS®Technology,whichenablesfastisolationofhighpurityandhighyieldexosomes.
Details
Backgroundinformation
Exosomesareextracellularvesiclesreleasedfromlivingcellsinanenergy-dependentprocess.Exosomesarespecificallyloadedwithnucleicacids,lipids,andproteinsfromtheirparentalcell.Therefore,theconstitutionofextracellularvesiclesreflectsthetypeandstatusoftheoriginatingcell.Exosomesaresecretedbymanycelltypes1intodiversebodyfluidssuchasblood,semen,urine,saliva,breastmilk,ascitesfluid,andcerebrospinalfluid.Themaindifferencetootherextracellularvesiclessuchasapoptoticvesiclesormembranevesiclesistheendocyticoriginofexosomes.Exosomesarereleasedfromintactcellsafterinwardbuddingofmultivesicularbodiesandfusionwiththeplasmamembrane.Theyhavethesamemembraneorientationastheoriginatingcell,i.e.,displayingextracellulardomainsontheirsurface.CD9,CD63,andCD81arethreeofthemost-studiedmembersofthetetraspaninproteinfamilyandcanbeusedtoisolateexosomes.
Detailedprocedure
Theisolationofexosomesorextracellularvesicles(EVs)isperformedbypositiveselectionusingMicroBeadsrecognizingthetetraspaninproteinsCD9,CD63,orCD81.First,EVsaremagneticallylabeledduringashortincubationperiod.ThelabeledEVsareloadedontoaµColumn,whichisplacedinthemagneticfieldofaµMACS™Separator.ThemagneticallylabeledEVsareretainedwithinthecolumn,whiletheunlabeledvesicelsandcellcomponentsrunthroughthecolumn.Afterremovingthecolumnfromthemagneticfield,theintactEVscaneitherbecollectedbyelutionwithIsolationBuffer,ordirectlylysedinthecolumnandtheproteininthelysatescanbeanalyzed,e.g.,byWesternblotting.