Dis$nguishing Ac$vated and Res$ng CD4+ T-Cell Populaons on … · 2017-08-28 · Dis$nguishing...
13
Dis$nguishing Ac$vated and Res$ng CD4+ T-Cell Popula$ons on the Moxi Flow Dylan Koundakjian Ryan Park Ragon Ins$tute of MGH, Harvard, & MIT
Transcript of Dis$nguishing Ac$vated and Res$ng CD4+ T-Cell Populaons on … · 2017-08-28 · Dis$nguishing...
Dis$nguishingAc$vatedandRes$ngCD4+T-CellPopula$onsontheMoxiFlow
DylanKoundakjianRyanPark
RagonIns$tuteofMGH,Harvard,&MIT
Res$ngCD4+
Diluted1:10inpropidiumiodide
Ac$vatedCD4+(Dynabeads)
Ac#vatedGateRes#ngGate
Res$ngandAc$vatedpopula$onsdis$nguishedbyCoulterprinciple(directvolumetricmeasurement)
LifeTechnologies
BothGates
ObservingCellDeathwithAn$bio$cs(Res$ngcellsonly)
UntreatedRes#ngCD4+ CD4+with2mg/mLZeocin
Notedecreasedlivecellcountandlargedeadcellpopula$oninZeocintreatedCD4+
ObservingCellDeathwithAn$bio$cs,FCSView(Res$ngcellsonly)
UntreatedRes#ngCD4+ CD4+with2mg/mLZeocin
Notedecreasedlivecellcountandlargedeadcellpopula$oninZeocintreatedCD4+
ObservingCellDeathwithAn$bio$cs(Res$ngandAc$vatedpopula$ons)Res#ngGate
CD4+with100ug/mLZeocin
Sizega$ngallowsobserva$onofdis$nctan$bio$ceffectsonAc$vatedandRes$ngpopula$ons
Ac#vatedGate
UntreatedCD4+
BothGates(FCSfiles)
UntreatedCD4+ CD4+with100ug/mLZeocin
Thisconcentra$onofZeocincausesintermediatecelldeathinRes$ngpopula$onandlargescaledeathinAc$vatedpopula$on
ObservingCellDeathwithAn$bio$cs(Res$ngandAc$vatedpopula$ons)Res#ngGate
CD4+with1ug/mLPuromycin(3Days)
Sizega$ngallowsobserva$onofdis$nctan$bio$ceffectsonAc$vatedandRes$ngpopula$ons
Ac#vatedGate
UntreatedCD4+(3Days)
BothGates(FCSfiles)
UntreatedCD4+ CD4+with1ug/mLPuromycin
Thisconcentra$onofcausestotaldeathinAc$vatedpopula$onwithlesseffectonRes$ngpopula$on
TrackingCD4+Ac$va$onOverConsecu$veDays(Untreated)
Res$ngGate
Ac$vatedGate
24hours 72hours48hours
Exponen$alincreaseoftheAc$vatedcellpopula$onisevidentfromMoxiFlowdata.
TrackingCD4+Ac$va$onOverConsecu$veDays(1ug/mLPuromycin)
Res$ngGate
Ac$vatedGate
24hours 72hours48hours
Noterela$vesurvivaloftheRes$ngpopula$onwhileAc$vatedpopula$ondecreasessignificantlyasaresultofPuromycinselec$on.
TrackingCD4+Ac$va$onOverConsecu$veDays(FCSview)
Untreated
1ug/mLPuromycin
24hours 72hours48hours
Noterela$vesurvivaloftheRes$ngpopula$onwhileAc$vatedpopula$ondecreasessignificantlyasaresultofPuromycinselec$on.
Conclusions• Moxiflowcanbeusedtodis$nguishRes$ngandAc$vatedCD4+popula$onsbyCoulterprinciple.
• TreatmentofCD4+cellswithdifferentan$bio$cshasdifferen$aleffectsonRes$ngandAc$vatedpopula$ons.
• Thesedifferen$aleffectscanbeobservedontheMoxiFlowthroughviabilitycoun$ngonspecificRes$ngandAc$vatedsizegates.
• Thesedataareusefulfordeterminingwhichan$bio$cstouseforselec$onindifferentscenariosinvolvingCD4+Tcells.
