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AAT Bioquest/MycoLight™ Ratiometric Bacterial Membrane Potential Kit *Red/Green Fluorescence*/22401/200 Tests
| Overview |  PrinterFriendlyVersion |
| Ex/Em(nm) | 484/520&630 |
| MW | N/A |
| CAS# | N/A |
| Solvent | DMSO |
| Storage | F/D/L |
| Category | MicroBIOLOGy FlowCytometry |
| Related | FluorescenceImaging |
| Spectrum | AdvancedSpectrumViewer |
Note1:Thawkitcomponentsatroomtemperaturebeforestartingyourexperiment.
Note2:TheKithasbeentestedatlogarithmicallygrowingculturesofthefollowingbacterialspecies:Micrococcusluteus,Staphylococcusaureus,S.warnerii,Bacilluscereus,Klebsiellapneumoniae,Escherichiacoli,andSalmonellacholeraesuis.
Note3:ManybacteriadonotshowaproportionalresponsetopartialmembranedepolarizationwithMycoStainIt™Green.Theresponseofeachbacterialsystemshouldbeinvestigatedandoptimized.OccasionallytheMycoStainIt™Greenconcentrationandstainingtimemustbeadjustedforoptimaldetectionofmembranepotential.Thefollowingistherecommendedprotocolforbacterialstaining.Theprotocolonlyprovidesaguideline,shouldbemodifiedaccordingtothespecificneeds.
Note4:Somecommonbuffercomponents,suchasTween20,sodiumazideandthimerosal,canaltermembranepotential,andshouldbeavoided.Besuretotestbufferadditivesfortheireffectonmembranepotentialduringoptimizationstudies.
1. Growbacteriainanyappropriatemedium.Bestresultsforhealthybacteriaareobtainedfromlog-phasecultures.Dilutethebacterialcultureto~106cellspermLinPBS(ComponentC)orequivalentsterilebuffer.Bacteriamaybediluteddirectlyfromtheculturemediumwithoutwashing.PreparesufficientsUSPensiontoprovide500µLpertest.
2. Aliquot500μlofthebacterialsuspensionintoaflowcytometrytubeforeachstainingexperimenttobeperformed.Preparetwoadditionaltubesforadepolarizedcontrolandanunstainedcontrol.
3. Add10μlof500μMCCCP(ComponentB)tothedepolarizedcontrolsampleandmix.
4. Add5μlof100XMycoStainIt™Green(ComponentA)toeachflowcytometrytubeandmix(donotaddstaintotheunstainedcontrolsample).Incubatesamplesatroomtemperaturefor30min.Stainedsamplescanbeanalyzedafter5min,butsignalintensitycontinuestoincreaseuntil~30min.
5. Stainedbacteriacanbeassayedinaflowcytometerequippedwithalaseremittingat488nm.Fluorescenceiscollectedinthegreen(fluoresceinfilter)andred(TexasRedfilter)channels. Theforwardscatter,sidescatter,andfluorescenceshouldbecollectedwithlogarithmicsignalamplification.
6. Instrumentadjustmentsareespeciallycriticalfordetectingrelativelysmallparticlessuchasbacteria.Usetheunstainedcontrolsampletolocatebacterialpopulationsintheforwardandsidescatterchannels.Usethesidescatterastheparameterforsettingtheacquisitiontrigger.
7. Applythedepolarizedcontrolsampleafteradjustingtheflowcytometerasdescribedabove.GateonbacteriausingforwardversussidescatterandadjustfluorescencephotomultipliertubevoltagessuchthatthegreenandredMFIvaluesareapproximatelyequal.Donotsetcompensation.
8. Whiletherelativeamountofredandgreenfluorescenceintensitywillvarywithcellsizeandaggregation,theratioofredtogreenfluorescenceintensitycanbeusedasasize-independentindicatorofmembranepotential.Thedatacanalsobeprocessedbygatingonbacteriausingforwardversussidescatter,andanalyzegatedpopulationswithadotplotofredversusgreenfluorescencereportingMFIvaluesaslinearvalues,notaschannels.
9. Onaratiometrichistogram,setMarkersaroundthepeaksofinterestandrecordthemeanratiovalues.Foradotplotofredversusgreenfluorescence,setregionsaroundthepopulationsofinterestandrecordredandgreenmeanfluorescenceintensity(MFI)valuesforeach.Toevaluatethedata,dividetheredpopulationMFIbythegreenpopulationMFI.
10. Intheflowcytometer,bacteriaareidentifiedsolelyonthebasisoftheirsizeandstainABIlity.Itisbesttoinspecteachsamplebyfluorescencemicroscopytoconfirmthattheparticlesdetectedareindeedbacteria.
| References&Citations |  PrinterFriendlyVersion |
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8. Lindback,T.;Rottenberg,M.E.;Roche,S.M.;Rorvik,L.M.,Theabilitytoenterintoanavirulentviablebutnon-culturable(VBNC)formiswidespreadamongListeriamonocytogenesisolatesfromsalmon,patientsandenvironment.VetRes2010,41(1),8.
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