BasicMethodforIndirectImmunofluorescenceLabeling

Background

Indirectlabelingismoreinvolvedthandirectlabeling.Ifyouneedtolabelcellswithdifferentantibodiesforsimultaneousanalysis,youmayencounterproblemschoosingsecondaryantibodies(Fluorochromeconjugatedantibodiesthatbindtotheprimaryantibody).Forexample,ifbothprimaryantibodiesaremouseIgG1,whatcanyoudotolabelthemindividuallywithdifferentfluorochromeconjugatedsecondaryantibodies?(FITC-conjugatedanti-MouseIgG1willlabelboth!)Thebiotin-avidinsystemmayprovideananswer,ifoneantibodycanbebiotinylated:

1. labelwiththenon-biotinylatedmouseantibodyfirst,
2. thentheFITCanti-mouseantibody,
3. thenthebiotinylatedmouseantibody,
4. thenPE-streptavidin.

OR,maybeoneoftheprimaryantibodiesisavailableinafluorochrome-conjugatedform;incubatethecellswithitaftertheprimaryandsecondaryantibodysteps.

Youcanseehowcomplicatedthiscanbecome...Thebestsolutionforsimultaneouslabelingisgetalldirectconjugates,ifatallpossIBLe.Thenseeourourprotocolfordirectlabeling.

Materials

1. PrimaryAntibodies:
   • Testantibody:UsuallymousemonoclonalIgGorIgM,notconjugatedwithanyfluorescentdyes.Forlabelingcellswithmorethanonetestantibodysimultaneously,youneedprimaryantibodiesmadeindifferentanimalsorhavedifferentIgsubclasses.Anotheroptionistohaveonofthemconjugatedtobiotin.

   • Negativecontrolsera:Purifiedprotein(usuallymouseIgMorIgGofthesamesubclass)tomatchyourtestantibodies.

     Inflowcytometry,fluorescenceisrelative.Weneedanegativecontroltodeterminewhere"positivity"begins.

   • Secondaryantibody:Fluorochrome-conjugatedantibodiesdirectedtowardstheproteincomposingtheprimaryantibodiesandnegativecontrolsera.

     Forsimultaneouslabeling,choosethefluorochromescarefully.Youwantthefarthestred-emittingantibodycombinationtolabeltheantigenwiththegreatestdensity(mostreceptorspercell).

   • Fluorochrome-conjugatedstreptavidin:Usethisifoneofyourprimaryantibodiesisbiotinconjugated.

   • PBSwith0.1%sodiumazideadded.Thesodiumazideassistsinpreventingcappingandsheddingorinternalizationoftheantibody-antigencomplexaftertheantibodiesbindtothereceptors.

   • CellsinsUSPension,countedandviABIlity-checked.Keeptheminculturemediumsupplementedwithantibioticsand2-5%fetalbovineserum,onice.Ifviabilityislessthan90%,consideraddinganotherfluorochrometoidentifydeadcellsduringanalysis.

   Equipment

   1. Centrifuge.YoushouldknowhowtheRPMtranslatesintoG-force.

   2. Precisionadjustablemicropipet.Youwillprobablyneedtwo:oneintherangeof10-100microliters,andanotherrangingfrom100-1000microliters.

   3. Vortexmixer.Youcouldmixbytappingorshakingthetubes,butamixerwillgivemuchmorereproducibleresultsinmostcases.

   4. 12x75mmpolystyrenetubes.Theclearplastickind.Ifyoucan,buytheFalconbrandbecausetheyfittheinstrumentbest.Ifyoucan"t,don"tworry-wewillsupplythemwhenyoubringyoursamplestothelab.

   5. Icebucketwithcover.Generally,cellsaremorestableandtolerateinsultbetterwhenthey"recold.Thecoverkeepslightout,whichcouldbleachthefluorochromes.

   6. Flowcytometer.Ifyouanalyzeyoursamplesinourlab,theinstrumentyouusewillmostlikelybeaFACScanorFACSort,madebyBecton-Dickinson.(Seeourinstrumentlistformoredetails.)

      Procedure

      1. Adjustthecellconcentrationto1millionperml.withculturemediaorPBS.

      2. Place1ml.ofthecellsuspensionintoeachofthe12x75tubes.

         Youwillneedatubeforeachprimaryandsecondaryantibodycombinationplusthenegativecontrol.Ifyou"redoingsimultaneouslabeling,useonetubeforeachcombinationofprimaryantibodiesorcontrols,butwewillneedsingle-antibodylabeledcellsforeachcombinationaswell.Confused?Let"stalk!

      3. Centrifugeat250xgfor5minutes.Useapipettoremovetheliquid.Becarefulnottodisturbthepellet.Aslightamountofliquidcanremain.

         Thisforceandtimeworkswellforlymphoidcells.Youmayhavetoadjustasrequiredifyourcellsaredifferent.

      4. Addtheappropriateamountofprimaryantibodyornegative-controlsera.Theamountisusuallygivenbythemanufacturer.Ifnot,itshouldhavebeendeterminedpreviouslybytitration,usingtargetcellswithalargenumberofreceptors.

      5. Vortex.Keepthetubesoniceforaround30minutes.Covertheicebucket.

      6. Firstwash-Add1ml.ofthePBS+azide.Vortex.

      7. Centrifugeandremoveliquidasabove.

      8. Secondwash-Add1ml.ofthePBS+azide.Vortex.

      9. Centrifugeandremoveliquidasabove.

      10. Addtheappropriateamountofsecondaryantibodyorfluorochrome-conjugatedstreptavidin.Theamountisusuallygivenbythemanufacturer.Ifnot,itshouldhavebeendeterminedpreviouslybytitration,usingtargetcellswithalargenumberofreceptors.

      11. Vortex.Keepthetubesoniceforaround30minutes.Covertheicebucket.

      12. Firstwash-Add1ml.ofthePBS+azide.Vortex.

      13. Centrifugeandremoveliquidasabove.

      14. Secondwash-Add1ml.ofthePBS+azide.Vortex.

      15. Centrifugeandremoveliquidasabove.

      16. Add1ml.ofthePBS+azide.Vortex.
      Keepthecellsonice,covered,untilyourscheduledtimeontheflowcytometer.Lymphoidcellswillusuallylastforseveralhours,thoughit"snotrecommendedtowaitthatlong.Ifyouanticipatewaitinglonger,considerfixingthecells,whichcanpreservethemforatleastseveraldaysoroftenlonger.