Comprehensive Analysis of Treg Subsets

Cryopreserved PBMC and/or single cell tumor samples were thawed and Treg subsets were assessed by flow cytometry as described before [19 (link)]. Antibodies and kits used were V500-labeled CD3 (clone UCHT1, BD Biosciences), AF700- or PE-Cy7-labeled CD4 (clone RPA-T4 and SK3 respectively, BD Biosciences), PE-CY7- or BV421-labeled CD25 (clone 2A3, BD Biosciences), BV650-labeled CD127 (clone A019D5, Biolegend), APC-H7-labeled CD45RA (clone HI100, BD Biosciences), PerCP-Cy5.5-labeled CD8 (clone SK1, BD Biosciences), PE-CF594- or Alexa Fluor 647-labeled Foxp3 (clone 259D/C7, BD Biosciences), BV421-labeled CTLA-4 (clone BNI3, BD Biosciences), FITC-labeled Ki67 (clone 20Raj1; eBiosciences), APC-labeled Helios (clone 22F6, Biolegend), PE-labeled Tbet (clone ebio4B10; eBiosciences), FITC-labeled CD14 (clone M5E2; BD Biosciences), LIVE-DEAD® Fixable yellow or near-IR dead cell stain kit (ThermoFisher Scientific), and the BD Transcription Factor Buffer set. Acquisition of cells was performed on a BD LSR Fortessa and flow cytometric sorting was done using a BD FACS Aria II. Data was analysed using DIVA software (version 8.02; BD Biosciences).

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Santegoets S.J., Duurland C.L., Jordanova E.S., van Ham J.J., Ehsan I., van Egmond S.L., Welters M.J, & van der Burg S.H. (2019). Tbet-positive regulatory T cells accumulate in oropharyngeal cancers with ongoing tumor-specific type 1 T cell responses. Journal for Immunotherapy of Cancer, 7, 14.

Publication 2019

LIVE-DEAD® Fixable yellow or near-IR dead cell stain kit BD LSR Fortessa BD FACS Aria II DIVA software

Alexa fluor 647 Antibodies Aria Buffer Cd25 Cells Clone Ctla 4 Cy5 5 Fitc Flow cytometric Stain Transcription factor Tumor

Corresponding Organization :

Other organizations : Leiden University Medical Center, Vrije Universiteit Amsterdam, Amsterdam University Medical Centers

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Treg subsets assessed by flow cytometry

control variables

Cryopreserved PBMC and/or single cell tumor samples
Antibodies and kits used for flow cytometry (V500-labeled CD3, AF700- or PE-Cy7-labeled CD4, PE-CY7- or BV421-labeled CD25, BV650-labeled CD127, APC-H7-labeled CD45RA, PerCP-Cy5.5-labeled CD8, PE-CF594- or Alexa Fluor 647-labeled Foxp3, BV421-labeled CTLA-4, FITC-labeled Ki67, APC-labeled Helios, PE-labeled Tbet, FITC-labeled CD14, LIVE-DEAD® Fixable yellow or near-IR dead cell stain kit, BD Transcription Factor Buffer set)
Flow cytometry acquisition on BD LSR Fortessa and sorting on BD FACS Aria II
Data analysis using DIVA software (version 8.02; BD Biosciences)

positive controls

None explicitly mentioned

negative controls

None explicitly mentioned

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