F4 80 bv421

Flow cytometry analysis was performed to examine the surface marker changes in THP-1 cells or BMDMs. THP-1 cells or BMDMs were seeded in a 6-well plate and treated with the respective compounds. Then, the cells were harvested with a cell scraper and washed with PBS three times. The acquired cells were blocked with CD16/32 antibodies (Clone 93, Biolegend) for 15 min on ice, and then PE-CD11b (Clone ICRF44, Biolegend), FITC-CD14 (Clone 63D3, Biolegend), BV421-F4/80 (Clone BM8, Biolegend), PE-Dazzle594-CCR7 (Clone 4B12, Biolegend), and PE-MMR (Clone C068C2, Biolegend) antibodies were applied to stain the cells for 30 min in the dark. Following washing in PBS, cell marker expression was assayed using FACS AriaIII (BD, New Jersey, USA), and the data were analyzed by FlowJo software (Tree Star Inc., San Carlos, USA).

Cell suspensions were stained with appropriate antibodies for 30 min on ice. The commercial antibodies used in this study included anti-mouse FITC-CD45 (BioLegend, CA, USA, #103108; 1 µg per 10⁶ cells), PE-CLEC2 (BioLegend, CA, USA, #146104; 1 µg per 10⁶ cells), BV421-F4/80 (BioLegend, CA, USA, #123137; 1 µg/10⁶ cells), Pe/Cy7-CD64 (BioLegend, CA, USA, #128016; 1 µg per 10⁶ cells), Percp/Cy5.5-CD11b (BioLegend, CA, USA, #101228; 1 µg per 10⁶ cells), APC/Cy7-PirB (R&D, CA, USA, #FAB2754S; 5 µg per 10⁶ cells), APC-Ly6C (BioLegend, CA, USA, #128016; 1 µg per 10⁶ cells), APC-Ly6G/Ly-6C (Gr-1) (BioLegend, CA, USA, #108411; 1 µg per 10⁶ cells), PerCP-CD19 (BioLegend, CA, USA, #115531; 1 µg per 10⁶ cells), APC-CD3 (BioLegend, CA, USA, #100235; 1 µg per 10⁶ cells), APC-CD206 (BioLegend, CA, USA, #141707; 2 µg per 10⁶ cells), and FITC-CD11c BioLegend, CA, USA, #117305; 1 µg per 10⁶ cells); and anti-human PE-CD14 (BD Biosciences Pharmingen, USA, #555398; 20 µl per 10⁶ cells), Mouse anti-human LILRB2 (R&D, R&D Systems, MN, USA, #MAB2078; 0.25 µg per 106 cells), and Goat Anti-Mouse FITC-IgG (Servicebio, Wuhan, China, #SF131; 1:200 dilution). All antibodies were diluted according to the manual from the manufacturer’s website. Dead cells and doublets were removed by dead-cell dye staining (Zombie Aqua Fixable Viability Kit, BioLegend, CA, USA, #B297827).

Tissue was homogenized in serum-free DMEM with 2 mg/ml collagenase type I C (VWR 234153-100MG) and incubated for 1 hour at 37°C. Following incubation, the sample was strained through a 45-μm filter and spun down for 5 minutes at 700 g. Cells were then stained with the following antibodies from BioLegend (1:300 in fluorescence-activated cell sorting [FACS] buffer) for 10 minutes: Alexa-488 CD326 (118210), BV-421 F4/80 (123131), BV-605 CD4 (100547), BV-510 cd11b (101245), Alexa-700 Ly6G (127622), APC CD25 (102012), PE/Cy7 cd11c (117317), APC/Cy7 CD45 (103116), and PE CD45RB (103308). Cells were analyzed on a 5-laser LSR II (Becton Dickson). Details of markers used to quantify individual cells types are presented in S2 Fig.

1 × 10⁶ cells from spleen, blood, mesenteric lymph nodules and colon tissue were stained with BV421-F4/80, APC-TLR2 and PECy7-PDL1 antibodies (Biolegend^®, San Diego, CA, USA) and subsequently incubated for 30 min at 4 °C in the dark. The cells were washed twice with 1 mL of FACS Sheat solution (Becton Dickinson, San Jose, CA, USA) and centrifuged at 1800 rpm for 5 min. The supernatant was decanted and the cells were resuspended in 350 µL of FACS Sheat (Becton Dickinson, San Jose, CA, USA). The cells were analyzed on the Attune NxT flow cytometer (ThermoFisher^®, Rockford, IL, USA) 10,000 events gated in the cell population of interest per sample were captured. Data analysis was performed with FlowJo software V X (Tree Star).