MAGPIX应用CCD成像原理取代200型的流式原理

MAGPIXChamberCapacityLuminexFlowCytometryvsMAGPIXInLuminexflowcytometersbeadsareinjectedthroughthecuvetteinasinglefile,countingandclassifyingbeadsastheypassby.Whenthebeadcountforallregionsreachesthedesiredvalue,thesystemstopsacquisitionandmovesontothenextwell.IntheMAGPIXsystemtheentireSampleLoadVolumeisinjectedintothechamberandthenimagesofthebeadsaretaken.Imageprocessingisperformed,andallthedataispresentedtotheuser.MAGPIXBeadLoadingTheMAGPIXchamberhasanupperendcapacityof~60,000-65,000beads.SincetheMAGPIXloadsallthebeadsintheSampleLoadVolumeintothechamber,thislimitsthetotalbeadsintheSampleLoadVolumetothesameupperlimit.Asageneralrule,theendusershouldtargetafinalcountof1,250beadsperregionintheSampleLoadVolume.(seeSampleLoadExamplesbelow)Atlowerbeadconcentrations,theTotalClassifiedEventsincreaselinearlywiththenumberofbeadsintheSampleLoadVolume.Asthebeadconcentrationloadedintothechamberincreases,theimageprocessingalgorithmsstarttothrowawaybeadsthatareinterferingwitheachother,andtheTotalClassifiedEventsdonotincreaseasfastastheconcentration.Asthebeadconcentrationapproachesthechambercapacity,theTotalClassifiedEventsstopincreasingwithincreasedbeadconcentration.IfthechamberisoverloadedtheTotalClassifiedEventswilldecrease.FlowCytometryandImagingComparisonIdentifybeadregionbasedoninternaldyeconcentrationsQuantifybindingeventsInterrogatebeadwithredlaser(635nm)Interrogatelabelwithgreenlaser(525nm)SheathFluidFlowCytometry-BasedAnalysisLED/Image-BasedAnalysisInterrogatelabelwithgreenLED(525nm)InterrogatebeadwithredLED(635nm)IdentifyandquantifywithCCDimagerBeadsinChamberMagneticCaptureTotalnumberofbeadsloadedinthechamber(FinalBeadconcentrationxSamplevolumeuptake)TotalClassifiedEventsMultiplexequivalent(assumingatargetloadof1,250beadsforallregions)ChamberloadingrangeChamberOverload6Plex50PlexLinearRange7,500beads62,500beads13,000ChamberCapacityClassificationCapacityTheMAGPIXsystempushestheentireSampleLoadVolumeintothechamber.Thus,theimagescanhavetoomanybeadsinthemtogiveaccurateresults.Theimageprocessingalgorithmsfavorthebrighterbeadsoverthedimmerbeads.InhigherPlexassays,thelowerintensityregionswillhavefewcountsthenthehighintensityregions.Asthechamberbecomesoverloaded,theuserwillseetheDotPlotstarttodropinthelowestCL1andCL2regionsfirst.Belowaretwo50-PlexDotPlots.Theoneonthelefthas1,250beadsperregionintheSampleLoadVolume.Theoneontherighthas3,750beadsperregionintheSampleLoadVolume(threetimesthenumbersuggested).Ingeneral,ifallthebeadsinyourmixareatthesameconcentration,thentheratiooftheregionwiththehighestcountstotheregionwiththelowestcountsshouldbeapproximately5orless(MaxCount/MinCount≤5).Iftheratioishigherthan5,itislikelythatthechamberisoverloaded.Theuserisabletoeffectthenumberofbeadsinthesampleloadbyadjustingtheinitialbeadconcentration,thebeadlossduetowashingsteps,thefinalresuspentionvolume,andtheSampleLoadVolumepickedupfromthewellandloadedintothechamber(seefigureonnextpage).WhentroubleshootingoverloadedDotPlots,thefirstparametertochangeistheSampleLoadVolume.TheSampleLoadVolumeissetto50uLbydefault.Itcanbevariedfrom20-200uL.BydecreasingtheSampleLoadVolumeyoudecreasethenumberofbeadsloadedintothechamber.Similarly,ifyouexperiencelowerthendesiredbeadcountsinalltheregions,thentheSampleLoadVolumecanbeincreasedtoincreasethenumberofbeadsloadedintothechamber.IfsettingtheSampleLoadVolumetotheminimumvalueof20uLisstilltoomanybeadsinthechamber,thentheresuspensionvolumeinthelaststepoftheassaycanbeincreasedtodecreasethebeadconcentration.Iftheresuspentionvolumecannotbechanged,thentheinitialbeadconcentrationcanbeadjusted.Thefinalre-suspensionvolumeshouldnotbegreaterthan150ul.SampleLoadExamples:Example1:2,500beadsperregionfora25-Plexassay=62,500beadsFinalresuspensionvolumeof100uLSampleLoadVolumesetto50uL=½of62,500beads=31,250beads(1,250perregion)Example2:5,000beadsperregionfora25-Plexassay=125,000beadsFinalresuspensionvolumeof100uLSampleLoadVolumesetto25uL=¼of125,000beads=31,250beads(1,250perregion)Numberofbeadsaddedtothewell(perregion)FinalResuspensionVolumeBeadretentionefficiencyFinalBeadconcentration(perregion)InitialBeadconcentration(perregion)VolumeofbeadmixusedperwellSampleVolumeNumberofbeadsloadedinthechamber1250Beads(perregion)Guaranteeatleast50beadcountperregionTargetload(perregion)TargetCount(perregion)SuccessfulAssayControlledbyassayprotocolControlledinXponentControlledbyoperatorandwashmethod12Orderofsuggestedchangestoimprovebeadcount:1:SampleVolume2:Finalresuspensionvolume3:Initialbeadconcentration3Finalbeadconcentration:TargetLoadOverloadOverload2500beads/region/100uLSampleVolume50µL100µL150µLNumberofbeadsloaded(perregion)1,2502,5003,750Totalnumberofbeadsloaded(fora50plex)62,500125,000187,5005000beads/region/100uLSampleVolume25µL50µL75µLNumberofbeadsloaded(perregion)1,2502,5003,750Totalnumberofbeadsloaded(fora50plex)62,500125,000187,500UnderloadPossibleunderloadTargetLoadPossibleoverloadOverloadFinalBeadConcentration:2500Beads/region/100µLSampleVolume10µL20µL25µL37µL50µL75µL100µL150µLNumberofbeadsloadedperregion2505006259251,2501,875