Cd14 brilliant violet711

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.

For characterization of Tregs, PBMCs were incubated first with EMA and Gamunex™, followed by indirect staining for CD3 with a primary CD3 antibody (OKT3 supernatant) and a Pacific Orange-conjugated secondary antibody (Invitrogen). After blocking the non-specific binding of the secondary antibody with mouse serum (Merck Millipore, Darmstadt, Germany), cells were directly stained with CD4-Pacific Blue, CD45RA-Alexa Fluor-700, CD8-peridinin-chlorophyll protein (PerCP), CD279-PerCP-Cy5.5, CD127-Alexa Fluor-647 (BioLegend) and CD25-APC-Cy7 (BD Biosciences). The cells were then fixed and permeabilized using the human FoxP3 kit (BioLegend) and the cells were stained for intracellular FoxP3 using a PE-conjugated antibody (BioLegend) according to the manufacturer’s instructions.
For characterization of MDSCs, PBMCs were stained with a cocktail of lineage (Lin) markers (CD3, CD19, CD56)-Brilliant Violet 605 (BioLegend, BD Biosciences), CD14-Brilliant Violet 711 (BioLegend), CD45-V500, CD15-FITC, HLA-DR PerCP-Cy5.5, CD11b APC-Cy7, CD33 Alexa Fluor-700 (BD Biosciences), and CD124-APC (R&D Systems, Minneapolis, MN, USA). All samples were measured using a BD LSRII (BD Biosciences) immediately after staining.