Anti cd14 clone m5e2

Human PBMCs were stained with anti-CD14 (clone M5E2, Biolegend), anti-CD16 (clone 3G8, Biolegend), anti-CD4 (clone OKT4, Biolegend), human CD39 (clone 498403, R&D Systems), human CD73 (clone 606112, R&D Systems)^{44 (link),45 (link)}, human CD25 (clone BC96, Biolegend), human FoxP3 (clone 236 A/E7, Biolegend), and human PD-1 (clone EH12.2H7, Biolegend). Prior to anti-FoxP3 staining, the cells were fixed and permeabilized.
Stained cells were analyzed in the Oklahoma Medical Research Facility (OMRF) Flow Cytometry Core Facility on a BD LSRII (BD Biosciences) and data was analyzed using FlowJo Software (Tree Star, Inc., Ashland, OR). Single stained samples were used to determine compensation values, and fluorescence minus one (FMO) controls were used to determine gating placement.

Cells were washed 2 times with cold PBS, scraped and collected by centrifugation and resuspended in FACS buffer (PBS supplemented with 0,5% bovine serum albumin and 5mM of EDTA). Cells were stained with anti-CD11b (clone ICRF44, FITC conjugated; Biolegend) or isotype control (mouse IgG1 FITC conjugated biolegend);, anti-CD14 (clone M5e2, APC conjugated; Biolegend) or isotype control (mouse, IgG2a, κ, APC conjugated, Biolegend), anti-CD206 (clone 15-2, APC/cy7 conjugated; Biolegend) or isotype control (mouse IgG1, APC/cy7 conjugated, Biolegend), anti-CD86 (clone IT2.2, Alexa Fluor 488 conjugated; Biolegend) or isotype control (mouse IgG2b, κ, Alexa Fluor 488 conjugated, Biolegend) and anti-CD163 (clone RM3/1, Alexa Fluor 647 conjugated; Biolegend) or isotype control (mouse IgG1, κ, Alexa Fluor 647 conjugated, Biolegend) Cells were analyzed on a BD LSRFortessa X-20 Cell Analyzer (BD Bioscience) with post-processing in FlowJo software (Tree star Inc). Cell populations were gated on forward and side scatter to select intact single cells. The gating strategy and a representative flow diagram is shown in S2 Fig.

Cells were analyzed by flow cytometry using the following anti-human antibodies: anti-CD14 (clone M5E2; BioLegend, London, UK), anti-CD86 (clone IT2.2; BioLegend), anti-CD83 (clone HB15e, BD Bioscience, Oxford, UK), anti-CD11c (clone 3.9; BioLegend), anti-CD1c (clone L161; BioLegend), anti-HLA-DR (clone, L243; BioLegend), anti-CD13 (clone WM15; BioLegend), anti-CD33 (clone P67.6; BioLegend), anti-CD141 (clone 1A4; BD Bioscience, Oxford, UK), and anti-CD11b (clone ICRF44; eBioscience). Dead cells were identified using fixable Viability dye Zombie UV (BioLegend, UK). The data were acquired on a LSRII flow cytometer or Fortessa (BD Bioscience) and analyzed using FACSDiva (BD Bioscience) or FlowJo software (Tree Star, Ashland, Oregon).