Dis$nguishing Ac$vated and Res$ng CD4+ T-Cell Populaons on … · 2017-08-28 · Dis$nguishing Ac$vated and Res$ng CD4+ T-Cell Populaons on the Moxi Flow Dylan Koundakjian Ryan Park

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.