Analysis and Sorting of T reg Analysis and Sorting of T reg Cells on the FACSAriaCells on the FACSAria

John DaleyJohn DaleyDirector of Flow CytometryDirector of Flow Cytometry

Hematologic NeoplasiaHematologic NeoplasiaDana-Farber Cancer InstituteDana-Farber Cancer Institute

Harvard Medical SchoolHarvard Medical School

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From BD Biosciences WebsiteFeatured products:Regulatory T cells

The way we used to sort Human The way we used to sort Human CD4+CD25+ PBL’s on the CD4+CD25+ PBL’s on the

FACSAriaFACSAria

SORT TOP2- 3% CD25 HI

Then CD45Ra was added to the Then CD45Ra was added to the Sort DesignSort Design

Four way reg sort

Multicolor(5) Minor populations : Four way sorting: Treg Story

CD45ra FITCCD127 PECD25 PE-CY5CD4 PE-CY7CD3 PAC BLUE

Compensation ComparisonsCompensation Comparisons

Manual Comp with cells and actual Manual Comp with cells and actual reagentsreagents

Autocomp with cells and actual reagentsAutocomp with cells and actual reagents

Autocomp with beads and actual reagentsAutocomp with beads and actual reagents

Autocomp with beads and CD45+ Autocomp with beads and CD45+ reagentsreagents

Autocomp with cells and CD45+ reagentsAutocomp with cells and CD45+ reagents

BD cells Cell Autocomp

BD cellsBead AutoComp

Treg Post Sort Reanalysis

99.4 97.198.4 99.9

POST SORT REANALYSIS: HUMAN PBL CD3+CD4+ CD45RA+ &CD45RA- Treg+/Teffector+/ --

Actual reagents cells compensation

cells

beads

Actual reagents beads compensation

cells beads

FoxP3 Expression in CD4+ Treg FoxP3 Expression in CD4+ Treg CD45RA+CD45RA+

Fluorescence minus 1Fluorescence minus 1