Analysis and Sorting of T reg Cells on the FACSAria
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Transcript of Analysis and Sorting of T reg Cells on the FACSAria
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 reagentsreagentsAutocomp with cells and actual reagentsAutocomp with cells and actual reagentsAutocomp with beads and actual reagentsAutocomp with beads and actual reagentsAutocomp with beads and CD45+ Autocomp with beads and CD45+ reagentsreagentsAutocomp 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