Cd14 pacific orange

Whole blood, PBMC, and nTreg staining was performed using the FoxP3 Staining Buffer Set purchased from eBioscience (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's recommendations. The following antibodies were used for wholeblood staining: CD14-PacificOrange (Invitrogen, Darmstadt, Germany); CD3-PacificBlue, CD4-AlexaFluor700, CD25-PE (BD Biosciences, Heidelberg, Germany); CD127-APC-AlexaFluor780, CD62L-PerCP-Cy5.5 (BioLegend, San Diego, CA); CD45RA-ECD (Beckman Coulter, Krefeld, Germany); and FoxP3-AlexaFluor488 (BD Pharmingen, Heidelberg, Germany). The following antibodies were used for PBMC stainings: CD3-PE-Cy7, CD4-AlexaFluor700, CD25-PE (BD Biosciences), CD14-PacificOrange (Invitrogen), CD127-APC-AlexaFluor780, CD45RA-ECD (Beckman Coulter); FoxP3-AlexaFluor488, CD137-PE-Cy5 (BD Biosciences), and CD154-PacificBlue (BioLegend). Dead cells were routinely detected by staining with aqua fluorescent reactive dye (Invitrogen). Data acquisition was performed on a FACS LSRII (BD Biosciences) or Navios flow cytometer (Beckman Coulter). FlowJo software V8 (Treestar) was used for further analysis. c l i n i c a l i n v e s t i g a t i o n S Landwehr-Kenzel et al.: nTregs in kidney transplantation nTregs were defined as CD4 þ CD25 high FoxP3 þ cells. By Boolean gating, non-Treg cells were identified and considered Tconv.