Anti cd14 pe cy7

T cells from healthy blood donors were co-cultured at 0.25×10⁶ cells/mL with monocytes from a subset of cirrhotic patients (N=9) or healthy controls (N=9) in round-bottom ultra-low attachment 96-well microplates (7007, Corning). Cells were incubated overnight at 37°C and 5% CO₂, and subsequently stained with an antibody panel comprising Live/Dead™ Yellow (Life Technologies), anti-CD14-PE-Cy7 (clone M5E2; BD Biosciences) or anti-CD14-FITC (clone: MφP9; BD Biosciences) and anti-CD3-FITC (clone: HIT3a; BD Biosciences) or anti-CD3-BUV496 (clone: UCHT1; BD Biosciences), and analysed on a five-laser BD LSRFortessa (BD Biosciences).

Briefly, PBMCs were washed and pelleted. Each PMBC sample was labelled with an antibody mix (anti-CD16-FITC, anti-CD25-PE, anti-CD14-PE-Cy7, anti-CD19-APC, and anti-CD3-APC-Cy7, all from BD, and 7-aminoactinomycin D from Life Technologies) and incubated for 20 min. Next, cells were washed and acquired in a Guava EasyCyte 8HT flow cytometer (Millipore, Merk, Madrid, Spain). Single staining and isotype controls were used to set the gating strategy. The percentage of each cell population of PBMCs (T, B, NK cells, and monocytes), their activation, and cell viability were analyzed. All conditions were studied in duplicate and the experiment was repeated twice.

One hundred microliters of whole blood were stained with anti-CD3-PECy7 (BioLegend, San Diego, CA, USA; clone HIT3a), anti-CD8-PECy5 (BioLegend; clone SK1), anti-PD-L1-PE (BioLegend; clone 29E.2A3), anti-CD14-PECy7 (BD Bioscience, San Jose, CA, USA; clone M5E2), anti-CD41a-FITC (Immunotools, Friesoythe, Germany; clone HIP8), and anti-CD62P-APC (Immunotools; clone HI62P). The cells were then incubated for 15 min in the dark before adding 2 mL of BD FACS lysing solution 1X (BD Bioscience) for 10 min. Finally, the cells were washed with 2 mL of PBS 1X and resuspended in 400 µL of PBS 1X before acquisition by flow cytometry (MACSQuant Analyzer 10 flow cytometer; Miltenyi Biotec, Bergisch Galdbach, Germany). Negative gates for each marker were defined by fluorescence minus one (FMO) controls.

Cryopreserved PBMCs were thawed, and dead cells were stained using the Live/Dead Fixable Cell Stain kit (Invitrogen, Carlsbad, CA). Cells were stained with fluorochrome-conjugated antibodies, including anti-CD3 (BV605; BD Biosciences), anti-CD4 (BV510; BD Biosciences), anti-CD8 (BV421; BD Biosciences), anti-CD14 (PE-Cy7; BD Biosciences), anti-CD19 (Alexa Fluor 700; BD Biosciences), and anti-CD56 (VioBright FITC; Miltenyi Biotec). For staining with anti-granzyme B (BD Biosciences), cells were permeabilized using a Foxp3 staining buffer kit (eBioscience).
For intracellular cytokine staining of IFN-γ, PBMCs were stimulated with phorbol 12-myristate 13-acetate (PMA, 50 ng/ml) (Sigma Aldrich) and ionomycin (1 μg/ml) (Sigma Aldrich). Brefeldin A (GolgiPlug, BD Biosciences) and monesin (GolgiStop, BD Biosciences) were added 1 hour later. After another 5 hours of incubation, cells were harvested for staining with the Live/Dead Fixable Cell Stain kit, anti-CD3, anti-CD4, and anti-CD8. Following cell permeabilization, cells were further stained with anti-IFN-γ (Alexa Fluor 488; eBioscience).
Flow cytometry was performed on an LSR II instrument using FACSDiva software (BD Biosciences) and the data analyzed using FlowJo software (Treestar, San Carlos, CA).

Images of monocytes, imMo-DCs, and mMo-DCs were captured by EVOS M5000 light microscope with phase contrast (Thermo Fisher Scientific). Cell recovery and viability were analyzed by NucleoCounter NC200 automated cell counter (ChemoMetec). ImMo-DCs and mMo-DCs were labeled with fluorochrome-conjugated monoclonal antibodies for 30 min at 4°C after blocking nonspecific sites with Human BD Fc Block (BD Biosciences) to detect expression of maturation markers and co-stimulatory molecules. The following mAbs were used: anti-CD14-PECy7, anti-CD80-PE, anti-CD83-BV786, anti-CD86-APC, anti-MHC class II antigen-BB515 (BD Biosciences), anti-CD1a-eFluor450 (Thermo Fisher Scientific). Nonspecific antibody binding was assessed using appropriate isotype controls (mouse IgG2a PE-Cy7, mouse IgG2a PE, mouse IgG1 BV786, mouse IgG1 APC, mouse IgG2a BB515, mouse IgG1 eFluor450). Cell debris and dead cells were excluded from the analysis based on light-scatter properties and 7-AAD fluorescence signal (BD Biosciences). Immunophenotype (IPT) was assessed by BD FACSCelesta SORP (BD Biosciences), and data analysis was carried out by FlowJo software (BD Biosciences).

For MDSCs characterization the following mAbs were used: anti-CD14/Pe-Cy7, anti-CD11b/APC-Cy7, anti-HLA-DR/PerCP-Cy5.5, anti-Lineage cocktail 1/FITC (BD Biosciences), anti-CD33/PE, anti-CD15/APC, anti-TGF-β/FITC (BioLegend) and anti-ARG1/AlexaFluor488, anti-IDO/AlexaFluor488, anti-IL-10/FITC (R&D Systems). For intracellular staining, the cells were fixed/permeabilized using a Cytofix/Cytoperm Fixation/Permeabilisation Kit (BD Biosciences, USA). Nonspecific staining was prevented by using FcR Blocker (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were analyzed with BD FACSCanto flow cytometer using FACS DIVA software (BD Biosciences, USA) and FCS express (De Novo Software, Los Angeles, CA). The gating strategy for MDSCs is shown in Figure 1A.