Analysis and Sorting of T reg Cells on the FACSAria

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Analysis and Sorting Analysis and Sorting of T reg Cells on the of T reg Cells on the FACSAria FACSAria John Daley John Daley Director of Flow Cytometry Director of Flow Cytometry Hematologic Neoplasia Hematologic Neoplasia Dana-Farber Cancer Institute Dana-Farber Cancer Institute Harvard Medical School Harvard Medical School tp://www.bdbiosciences.com/features/products Picture credit:

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Analysis and Sorting of T reg Cells on the FACSAria. John Daley Director of Flow Cytometry Hematologic Neoplasia Dana-Farber Cancer Institute Harvard Medical School. Picture credit:. http://www.bdbiosciences.com/features/products. From BD Biosciences Website Featured products: - PowerPoint PPT Presentation

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

Page 1: 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

http://www.bdbiosciences.com/features/products

Picture credit:

Page 2: Analysis and Sorting of T reg Cells on the FACSAria

http://www.bdbiosciences.com/features/products/display_product.php?keyID=162

From BD Biosciences WebsiteFeatured products:Regulatory T cells

Page 3: Analysis and Sorting of T reg Cells on the FACSAria

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

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

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Four way reg sort

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

CD45ra FITCCD127 PECD25 PE-CY5CD4 PE-CY7CD3 PAC BLUE

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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

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BD cells Cell Autocomp

BD cellsBead AutoComp

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Treg Post Sort Reanalysis

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99.4 97.198.4 99.9

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

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Actual reagents cells compensation

cells

beads

Actual reagents beads compensation

cells beads

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

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Fluorescence minus 1Fluorescence minus 1