Cd127 pe clone hil 7r m21

Peripheral blood mononuclear cells and subcortical white matter‐derived single cell fractions were isolated after rapid post‐mortem autopsies of NBB brain donors as described previously 56, 57. Donor and sample characteristics are listed in Table S7. Cells were stained with fixable viability dye eFluor 780 (Life Technologies) and the following antibodies: CD3 PE‐Cy5.5, clone SK7, (Invitrogen), CD20 APC, clone L27, CD25 FITC, clone 2A3, CD69 BV395, clone FN50, CD127 PE, clone HIL‐7R‐M21, (BD Biosciences), CD4 BV510, clone RPA‐T4, CD8a BV785, clone RPA‐T8, and FAS PE‐Cy7, clone DX2, (Biolegend), and analyzed on a Fortessa LSR^TM cell analyzer (BD Biosciences, San Jose, CA, USA). Data were analyzed with FlowJo software 10.5 (Tree Star, Ashland, OR, USA).

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.