• Single-useand/orpipetteswithdisposabletips2-100μL
• Polypropylenetubes
• Pipettes1mLand5mLforreagentpreparation
• Deionizedwater
• PBS
• BSAorFBSorFCS
• Disposablepipettingreservoirs
• PreparetheWashingbuffer(notprovidedinthekit)
• FACStubes

Furtherdetailsregardingsampletype:Saliva

ExoFACS™AssayProtocol:
1. HumanSalivasamplepreparation:Add1XPBSinsalivasamplesinratio1/1(5 mLofsaliva+5 mLof1XPBS).Mixtogetherandproceedtothefollowingcentrifugationsteps:
   • 15 minat2600g.
   • 20 minat15,550g.filterthrough0.22umfilter.
2. ExosomeisolationfromSaliva:
   Fluid:Saliva
   Minimumvolumerequired:200 μL
   Volumesuggested:250 μL-500 μL
   • AddExoPure™solutiontoyoursampleinratio1/4.
   • Mixwellbypipettingandinvertingtube.
   • Incubateonicefor1hr.
   • Centrifuge20 minat10,000g(centrifugecanbeperformedat4 °CoratRT).
   • Discardthesupernatant.
   • Centrifugefor2 minat1500gtoeliminateentirelythesupernatant.
   • Resuspendthepelletin100 μL*of1XPBS.*Volumeofresuspensioncanbedefinedbytheuser.
3. LyophilizedExosomeStandardreconstitution:
   • Reconstitutelyophilizedexosomestandardbyadding100 μLofdeionizedwatertogetafinalconcentrationof1 μg/μl.
   • Resuspendexosomespipettingthesolutionupanddown10-15times,avoidingbubbles.
   • Vortexthereconstitutedstandardfor60secs.Brieflycentrifugethetubescontainingthestandardtoensurethatthesolutioniscollectedatthebottomofthetube.Pipettethesolutionupanddown10times,avoidingtheintroductionofbubbles.Afterthisstep,thestandardisreadytouse.
   • Use5 μLofreconstitutedExosomeStandardforeachreaction.
4. ExosomebindingontolatexFACS-Beads:
   • ItisrecommendedtopreparethecomplexExosome-Beads(Exo-Beads)inonesingletube,thentodivideinsinglereactionsbeforetheantibodyincubation.
   • LatexFACS-Beadsarereadytouse.ResuspendwellFACS-Beadspriortousebyvortexingorpipettingseveraltimes.
   • Foreachreactionmixtogether5 μLofExosomeStandardsand5 μLofFACS-beadsinaneppendorftube(preferablylowbinding).Mixwellbypipetting5-6times.Example:ifyouwanttorun10reactions,mixintothesameeppendorflowbindingtube50 μLofExosomeStandardsand25 μLofFACS-Beads.
   • ForexosomeisolatedbyExoPureTM,mix25 μLofFACS-Beadswiththevolumeofresuspendedexosomessuggested(volumesareindicativeonly,theusershoulddefinetheappropriatevolumesonthebaseofexosomeyield).
   • Incubatefor15 minatroomtemperature(RT).
   • Add0.7 mLof1XPBSandincubateinrotatororshakerfor2hratRTorovernight(ON)at4 °C.
   • CentrifugethecomplexExosomes-Beads(Exo-Beads)for5 minat4500gat4 °Canddiscardthesupernatant.
   • Add1 mLofSampleBuffer,resuspendExo-Beadsfor5-6timesandincubatefor15 minatRT.
   • Centrifugefor5 minat4500gat4 °C,discardthesupernatant.
5. AntibodyIncubation:
   • PreparetheWashingbuffer(notprovidedinthekit)diluting2 %ofFBS(orFCS)in1XPBS(considerthatyouneed8 mLofwashingbufferforeachreaction).Alternatively,ifyoudonthaveFBSorFCS,preparetheWashingbufferdiluting0.5 %ofBSAin1XPBS.Keeponice.
   • ResuspendtheExo-Beadsin100 μLofSamplebufferforeachreaction.Example:ifyouarerunning10reactionsresuspendExo-Beadsin1 mLofSamplebuffer.
   • PreparetheFACStubes(notprovidedinthekit),onetubeforeachreaction.
   • DividetheExo-BeadsresuspendedinsamplebufferineachFACStube,pipetting100 μLofsuspensionineachtube.
   • Addprimaryantibodyinratio1:200(0.5 μLpereachFACStube)*.*Ifotherprimaryantibodiesareusedthecorrectdilutionmustbedefinedbytheuser.
   • Incubatefor2hrat4 °Cinthedark).Fornegativecontrol,PEorFITC-anti-MouseIgG1isotypeorFITCorPEanti-RabbitIgG1canbeused.(isotypecontrolsnotprovidedinthekit)OtherwiseincubatecontrolsampleswithsecondaryAbsonly(eitherleaveiniceordirectlyaddsecAbs).
   • Add4 mLofpreparedWashingbuffertoeachFACStube.
   • Centrifuge5 minat4000ganddiscardthesupernatant.
   • ResuspendbeadspelletintotheFACStubesin100 μLofSamplebuffer.
   • Addsecondaryantibodyinratio1:2000.(mix57 μLofSamplebufferwith3 μLofsecondaryantibody,add1 μLofthereceivedsolutionpereachFACStubetoobtaintherightdilutionofantibody).
   • Incubatefor1hrinthedarkat4 °C.
   • Add4 mLofWashingbufferineachFACStube,centrifugefor5 minat4000gat4 °C.Discardsupernatantbypouringitout.VortexwhathasremainedinsidetheFACStubes.Ifnotuseimmediately,putinthedarkat4 °C.
   • Add500 μLofWashingbufferperFACStube.
   • Analyzethesamples.
   • Read10,000eventsfromthegatedfirstpopulation.
   • Alexa488isreadinFL1channel(green)
6. Reproducibility:ExoFACSTMisausefultoolforexosomeproteinprofilingbyusingFACStechnique.ExoFACSTMwasusedforaproteinmarkerprofileinexosomesderivedfromdifferentsources.ExosomebindingonFACS-beadswasperformedbyincubationat4 °Covernight.Exosome-beadcomplexisreadytobelabeledwithfluorophore-conjugatedantibodiesforspecificexosomemarkers.

• ExoFACS^TMcontainsreagentsandantibodiesfor20reactions.
• Exosomestandardsmustbereconstitutedin100μLofdeionizedwater.
• ExoPure™isincludedinthekitforexosomeisolation.
• Beadsarereadytouseforexosomecapture.
• Primaryandsecondaryantibodymustbeappropriatelydilutedinsamplebuffer.
• 1vial(100μg)ofExosomestandards(lyophilized),fromhumansaliva(numberofparticles/mL1x1010).
• Exosomestandards:Theremainingreconstitutedstandardstocksolutionshouldbealiquotedintopolypropylenevials(preferablylowbinding)andstoredat-20°Cforuptoonemonthorat-80°Cforuptosixmonths.Strictlyavoidrepeatedfreeze-and-thawcycles.
• Storeopenedanddilutedreagentsat4°Cupto12monthsifunopened.