Anti cd14 apc cy7 clone mφp 9

Cryo-recovered PBMC were cultured at 2.5×10⁶ cells/mL in presence of 0.1 μg/mL LPS (eBioscience), 1 μg/mL of the synthetic toll-like receptor agonist CL097 (In vivogen), or no stimuli in round-bottom 96-well microplates at 37°C and 5% CO₂ for five hours. Two patients with cirrhosis with insufficient cell numbers after cryo-recovery were not stimulated with LPS. After stimulation, supernatants were collected and cells were stained with a panel of antibodies comprising anti-CD1c (clone L161; BioLegend), anti-CD11b-PE (clone ICRF44; BD Biosciences), anti-CD16-BV605 (clone 3G8; BD Biosciences), anti-CD14-APC-Cy7 (clone MφP-9; BD Biosciences), anti-CD141-APC (clone 1A4; BD Biosciences), anti-CD33-PE-Cy7 (clone P67.6; BD Biosciences), anti-CD86-BV711 (clone 2331; BD Biosciences), anti-HLA-DR-BV786 (clone G46-6; BD Biosciences), anti-PD-L1-BUV395 (clone MIHI; BD Biosciences), anti-CD19-PerCP-Cy5.5 (clone SJ25C1; BD Biosciences), and Live/Dead™ Yellow (Life Technologies). Flow cytometric analysis was performed on a five-laser BD LSRFortessa (BD Biosciences). Gatings of cell populations were set based on an internal PBMC control that was run in each experiment.

Peripheral whole blood from human donors was collected into an acid citrate dextrose Vacutainer (BD Biosciences). Blood was transferred to a 50 ml conical tube and diluted 1:10 with ammonium chloride potassium lysis buffer (ThermoFisher Scientific) to lyse red blood cells. Following a gentle inversion to mix, the blood and lysis buffer were incubated together for 5 min at the room temperature. White blood cells were collected by centrifugation at 500 × g for 5 min. Cell pellet was resuspended and washed twice in cold phosphate buffered saline (PBS) and collected by centrifugation at 500 × g for 5 min followed by a third wash in complete growth medium (RPMI1640 containing 10% FCS, 2mM l-glutamine, 1% penicillin/streptomycin, and 1% HEPES). White blood cells were counted and resuspended to a final concentration of 5 × 10⁵ cells/ml in complete growth medium for immediate use in the phagocytosis assay. To identify phagocytosis by the neutrophils, the white blood cells were stained with anti-CD66b pacific blue (clone G10F5; BioLegend), anti-CD3 AlexaFluor 700 (clone UCHT1; BD Biosciences), and anti-CD14 APC-Cy7 (clone MφP9; BD Biosciences). Neutrophils were defined as positive for a high side scatter area (SSC-A^high), CD66b⁺, CD3⁻, and CD14⁻.