Anti cd14 fitc

To detect intracellular production of IFN-γ, PBMCs from patients and healthy donors were incubated overnight at 37°C with IL-12 (0.5 ng/ml), or IL-12 (0.5 ng/ml) and IL-18 (0.1 ng/ml) combined. Surface staining was done by incubating the cells with FITC anti-CD3, PC5 anti-CD56, FITC anti-CD14 (Beckman Coulter), FITC anti-CD20, and APC/Cy7 anti-CD16 (BD) mAbs for 30 min at 4°C. Cells were then washed, fixed, and permeabilized with BD Cytofix/Cytoperm kit (BD Biosciences Pharmingen). IFN-γ production was detected by subsequent intracellular staining with PE-conjugated anti-IFN-γ (BD Biosciences Pharmingen). After washing, the proportion of CD3⁻ CD14⁻ CD20⁻ CD56⁺ cells expressing IFN-γ was immediately analyzed on LSR Fortessa Flow Cytometer (BD) using FACSDiva software (BD). Final analysis was done using FloJo v.10.2 (TreeStar).

Five milliliters of venous peripheral blood from patients and healthy volunteers was collected in EDTA-coated tubes. Samples of 50 μL blood per tube were transferred for staining with monoclonal antibodies and red blood cell (RBC) lysis. Peripheral blood samples were stained according to the manufacturer's recommendations using the following fluorochrome-coupled antibodies: anti-CD14-FITC (IM0645U, Beckman Coulter Company), anti-HLA-DR-PB (A74781, Beckman Coulter Company), and anti-CD45-KO (A96416, Beckman Coulter Company). Whole blood cells were incubated with antibodies against surface markers at room temperature (approximate range 20°C to 25°C) for 15 min in the dark. After RBC lysis washing, cells were collected and analyzed with Navios Cytometer (Beckman Coulter Company). Mo-MDSCs were defined as CD45⁺, CD14⁺, and HLA-DR^low/−. Isotype-matched antibodies were used in all samples as controls. Specifically, the population defined as HLA-DR^low/− was based on isotype staining with gating to include, at a minimum, 95% of the CD14⁺ population.

Whole blood samples (100 μl) were stained with anti-CD3 ECD, anti-HLA-DR PE (or ECD) (Invitrogen), anti-CD4 PE (or ECD), anti-CD8 FITC, anti-CD19 FITC (or PE), anti-CD14 FITC (or PC5), anti-CD15 PE, anti-CD57 FITC (Beckman Coulter) and isotype controls (Beckman Coulter) for 25 min. at +4°C. Intracellular staining with anti-Foxp3 (eBioscience) was performed after surface staining with anti-CD4 PE, followed by fixation and permeabilisation with Foxp3-Transcription Factor Staining Buffer Set (eBioscience) according to the manufacturer’s recommendations.
Samples were acquired on a Cytomics FC500 (Beckman Coulter) and analyzed with Flowing Software (version 2.5.1; Turku Centre for Biotechnology, University of Turku, Finland [http://www.flowingsoftware.com/]).

EDTA whole blood samples from 5 healthy subjects, 10 patients with mild and 10 patients with severe disease were stained with anti-HLA-DR PE and ECD, anti-CD3 ECD, anti-CD4 PE, anti-CD8 FITC, anti-CD19 PC7, anti-CD14 FITC, anti-CD16 PC5, anti-CD15 PE, anti-CD57 FITC, anti-CD56 PE, anti-CD11c PCP, anti-CD123 PE, anti-CD83 PE, anti-CD38 PE, anti-CD23 ECD and isotype controls (all from Beckman Coulter) for 20 minutes in the dark at +4°C. Samples were analyzed on the flow cytometer Cytomics FC500 (Beckman Coulter). Data were processed by FlowJo V.10.

Blood was centrifuged and serum was stored in -80 °C until assayed. Levels of biomarkers were quantified by using commercially available kits of enzyme immunosorbent assays from Bio-Techne (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. The lower limits of detection were: 16 pg/ml for tumor necrosis factor-alpha (TNFα); 40 pg/ml for IL-6; 31 pg/ml for IL-10; 62 pg/ml for IL-38; 156 pg/ml for IFNγ; and 313 p/ml for platelet-derived growth factor (PDGF)-A.
White blood cells were incubated for 15 minutes in the dark with the monoclonal antibodies anti-CD14 FITC, anti-HLA-DR-PE, anti-CD45 PC5 (Beckman Coulter, Marseille, France). White blood cells were also incubated for 15 minutes in the dark with anti-CD3 FITC, anti-CD4 FITC and anti-CD19 FITC (fluorescein isothiocyanate, emission 525nm, Beckman Coulter); with anti-CD4 PE, anti-CD8 PE, and anti-CD(16+56) PE (phycoerythrin, emission 575nm, Beckman Coulter); and with anti-CD45 PC5 (emission 667nm, Beckman Coulter). Fluorospheres (Beckman Coulter) were used for the determination of absolute counts. Cells were analyzed after running through the CYTOMICS FC500 flow cytometer (Beckman Coulter Co, Miami, Florida). Isotypic IgG controls stained also with anti-CD45 were used for each patient. Results of HLA-DR on CD14/CD45-cells were expressed as mean fluorescence intensity (MFI).