Phenotypic analysis of Treg subsets

Cryopreserved PBMCs were thawed and stained with viability dye (eFluor 780; eBioscience). Antibodies against CD4 (FITC or V500), CD25 (APC), CD45RA (Alexa Fluor 700)(BD Biosciences), CD127 (PE-eFluor 610), FOXP3 (PE), TIGIT (PerCP-eFluor710) (eBioscience), Helios (Pacific Blue; Biolegend). Purified anti-FCRL3 antibody was provided by Nagata (37 (link)), and was detected with F(ab′)2 anti-mouse IgG (PE-Cy7; eBioscience). Flow cytometry analysis was performed on an LSR Fortessa analyzer (BD Biosciences), and sorting throughout this study was performed on a FACS Fusion cell sorter (BD Biosciences). Recombinant human IL-6, IL-27, CLC, IL-11 (R&D systems), and LIF (Peprotech) were added to suppression assays where indicated at the time of activation.

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Bin Dhuban K., Bartolucci S., d'Hennezel E, & Piccirillo C.A. (2019). Signaling Through gp130 Compromises Suppressive Function in Human FOXP3+ Regulatory T Cells. Frontiers in Immunology, 10, 1532.

Publication 2019

eFluor 780 PE-eFluor 610 TIGIT (PerCP-eFluor710) Helios (Pacific Blue; LSR Fortessa analyzer FACS Fusion cell sorter Recombinant human IL-6 IL-11

Anti antibody Anti igg Antibodies Assays Cd25 Cell fusion Fitc Flow cytometry Human Il 11 Il 27 Mouse Tigit

Corresponding Organization :

Other organizations : McGill University Health Centre, Translational Research in Oncology, McGill University

Top 5 similar protocols

Variable analysis

independent variables

Recombinant human IL-6
Recombinant human IL-27
Recombinant human CLC
Recombinant human IL-11
Recombinant human LIF

dependent variables

Immune cell population and function

control variables

Cryopreserved PBMCs
Viability dye (eFluor 780; eBioscience)
Antibodies against CD4 (FITC or V500), CD25 (APC), CD45RA (Alexa Fluor 700)(BD Biosciences), CD127 (PE-eFluor 610), FOXP3 (PE), TIGIT (PerCP-eFluor710) (eBioscience), Helios (Pacific Blue; Biolegend)
Purified anti-FCRL3 antibody detected with F(ab′)2 anti-mouse IgG (PE-Cy7; eBioscience)
Flow cytometry analysis on an LSR Fortessa analyzer (BD Biosciences)
Cell sorting on a FACS Fusion cell sorter (BD Biosciences)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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