Cd127 alexa fluor 647

For cell phenotype analysis, monoclonal antibodies were directly added to 100 μL aliquots of heparinized whole blood and incubated at room temperature for 15 min. The antibodies used were: CD3 PECy7, CD14 APC, CD56 PE-Cy5 and CD19 PerCp (Biolegend, San Diego, CA, USA), CD4 Viogreen (Miltenyi Biotec GmBh, Bergisch Gladbach, Germany), CD39 Fitc, CD73 PE, CD25 BV421 and CD127 AlexaFluor 647 (BD Biosciences, San Jose, CA, USA). Red blood cells were lysed and white cells were fixed using BD FACS lysing solution (BD Bioscience). Negative populations were determined using respective anti-human isotype controls. Samples were acquired using a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec GmBh) and the data were analyzed using FlowJo software v. 10.0 (TreeStar Inc., Ashland, OR, USA).
We compared the frequencies of four subsets based on CD39 and CD73 expression (CD39⁻CD73⁺, CD39⁺CD73⁺, CD39⁺CD73⁻, and CD39⁻CD73⁻) within each studied population (B cells as CD3⁻CD19⁺, CD4⁺ T cells as CD3⁺CD4⁺, CD8⁺ T cells as CD3⁺CD4⁻, NK cells as CD3⁻CD4⁻CD19⁻CD56⁺, Tregs as CD3⁺CD4⁺CD25⁺⁺CD127⁻, and monocytes as CD14⁺). The percentage of these four subsets was related to their corresponding precursor populations (CD4⁺ T, CD8⁺ T, B, NK, and monocytes). In the case of Treg, the percentage was related to CD4⁺ T cells.

PBMCs were thawed, washed and incubated with Gamunex and EMA, then stained with a cocktail of lineage (Lin) markers (CD3, CD19, CD56)-Brilliant Violet 605 (BioLegend, BD-Biosciences), CD14-Brilliant Violet711, CD11c-PE-Cy7 (BioLegend), CD45-V500, CD123-BV421, HLA-DR-PerCP-Cy5.5, CD15-FITC, CD11b-APC-Cy7, CD33-Alexa Fluor 700 (BD-Biosciences), and CD124-APC (R&D Systems) using EMA to identify dead cells.
To characterize Tregs, we used the same panel as before [23 (link)], staining PBMCs forCD3 (OKT3 supernatant) with Pacific Orange-conjugated secondary antibody (Invitrogen) followed by staining for CD4-Pacific Blue, CD45RA-Alexa Fluor-700, CD8-Peridinin-chlorophyll protein (PerCP), CD279-PerCP-Cy5.5, CD127-Alexa Fluor-647 (Bio legend), CD25-APC-Cy7 (BD Biosciences) and intracellular staining for FoxP3-PE (Bio legend). All samples were measured using a BD LSRII (BD Biosciences) immediately after staining.