Cd16 percp cy5

Cells were collected and washed once in buffer (PBS, 1% FBS (Biochrom), 0.5% Sodium azide (Carl-Roth)). The supernatant was discarded completely and 50 µL staining solution in buffer containing all labeling antibodies was added and incubated for 30 min at 4 °C. Subsequently, the suspension was washed once with buffer and suspended in buffer containing 1% Paraformaldehyde (PFA, Carl-Roth) and analyzed the next day. The following antibodies were used for macrophage staining: CD163-Fitc (1:20, BioLegend, San Diego, CA, USA), CD80-PE (1:20, BioLegend), CD16-PerCPCy5.5 (1:200, BioLegend), CD206-APC (1:100, BioLegend), CD14-APCCy7 (1:100, BD, Franklin Lakes, NJ, USA), HLA-DR-PeCy7 (1:400, BioLegend), Life/Dead-V510 (1:100, Thermo-Fisher). Following antibodies were used for T cell staining in the proliferation setup: CD8-PE (1:50, Miltenyi Biotec), CD4-APC (1:100, BioLegend), CD3-APCCy7 (1:100, BioLegend).

100μL of whole blood samples from HBV-related ACLF, patients with chronic hepatitis B (CHB) or healthy controls (HC) were pre-incubated with suPAR(50 ng/mL, R&D, USA) or PBS for 45 min at 37 °C in 5% CO₂. Next, all samples were incubated with heat-inactivated E. coli (8 × 10⁷ cfu/mL) in 96-well plates for 30 min. Then the cells were harvested for CD16-percp-cy5.5 (Biolegend, USA) staining and oxidative burst assessment using an ROS assay kit (Genecopoeia, MD, USA) and were analyzed by a LSRFortessa cytometer (BD bioscience, USA) according to the manufacturer’s instructions. Neutrophils were indicated as CD16⁺.

Frozen PBMCs were thawed rapidly, washed, and then stimulated with 10 μM R848 (Invitrogen, Carlsbad, CA) for 22 h [25 (link)]. Brefeldin A was added 10 h prior to the end of the incubation period. PBMCs were exposed to antibodies against lineage markers CD3-PerCP/CY5.5, CD19-PerCP/CY5.5, CD14-PerCP/CY5.5 (Biolegend, San Diego, CA), CD16-PerCP/CY5.5 (Biolegend, San Diego, CA), CD56-PerCP/CY5.5 (Biolegend, San Diego, CA), HLA-DR-APC/CY7, CD11c-PE/CY7 (eBioscience, San Diego, CA), CD1c-PE (Biolegend, San Diego, CA) for 30 min in the dark at 4 °C. Each tube receiving 250 μL of Cytofix/Cytoperm solution was vortexed gently, and incubated for 20 min in the dark at 4 °C. The samples were then washed, exposed to antibodies against IL-12, incubated for 30 min in the dark at 4 °C, and fixed with 1 % paraformaldehyde. At least 200,000 events were collected from each sample with BD FACS LSRII, and the data obtained were analyzed with FlowJo.

Purified human monocytes were thawed and cultured in complete RPMI at a density of 2 × 10⁶ cells per well of a 6-well plate containing 50 ng/mL macrophage colony-stimulating factor (M-CSF; Miltenyi Biotec) for 7 days. Differentiated macrophages (M0) were harvested with a cell scraper and washed with PBS. In 200 µL complete RPMI, 1 × 10⁵ cells were seeded directly onto the intimal surface of the CFC or IFC human aortic tissue punch which had been frozen and rewarmed as described above. To ensure direct contact of the macrophages with the tissue the punch was held to the bottom of the 48-well plate with a small ring cut from a silicone tube (Ismatec/Cole-Parmer, Wertheim, Germany). After 2-day co-cultures at 37 °C, macrophages were harvested from the tissue surface with Accutase (Gibco/Thermo Fisher Scientific) and stained with the human specific antibodies CD163-FITC (1:20), CD80-PE (1:20), CD16-PerCPCy5.5 (1:200), CD206-APC (1:100), HLA-DR-PECy7 (1:400) (all BioLegend), CD14-APCCy7 (1:100) (BD Biosciences) and a viability marker in the V450 channel (1:1000) (LIVE/DEAD® Fixable Violet Dead Cell Stain Kit; Invitrogen/Thermo Fisher Scientific). FACS staining and measurement was performed as described above.