Isolation of Regulatory and Conventional T Cells

Flow cytometry cell sorting was used for the isolation of CD4⁺CD127^loCD25^hi Treg from PBMCs, unaffected colon tissue and tumor and CD4⁺CD127⁺CD25⁻ conventional T cells from PBMCs and unaffected colon tissue [26 (link)]. Buffy coats from healthy volunteers were pre-enriched for CD4⁺ T cells via negative selection using immunomagnetic sorting with EasySep Human CD4⁺ T cell Isolation (Stemcell). Enriched CD4⁺ T cells were sorted using the following antibodies and reagents: Live/Dead fixable aqua dead cell stain kit from Molecular Probes; CD4-PerCP (clone OKT4) from BioLegend; CD8-BV711 (clone RPA-T8), CD25−BV650 (clone M-A251) and CD127-PE (clone HIL-7R-M21) from BD Biosciences; CD127-FITC (clone eBioRDR5) from eBioscience; and CD39−FITC (clone A1) from AbD Serotec, on a FACS Aria (BD Biosciences) equipped with FACS Diva software (BD Biosciences). FMO and isotype controls were used as reference to determine marker expression. The sorted cells were manually counted with Trypan Blue solution and mean viability of sorted cells was 94% for Treg and 92% for conventional T cells.