Cd14 bv785 clone m5e2

Liver cells were freshly isolated from liver tissue as described above. Two million liver cells were pelleted by centrifugation and resuspended in 100 uL of pHrodo E. coli bioparticles (Life Technologies, P35361) prepared at 1 mg/mL in PBS + 2% FBS. Cells were incubated with E. coli bioparticles for 2 hours at 37°C, 5% CO₂. A negative control was conducted using 10 uM cytochalasin D. Cells were washed once with PBS + 2% FBS and then stained with CD3-Pacific Blue clone SP34-2 (BD Biosciences), CD4-BV650 clone OKT4 (Biolegend), CD8-APC-H7 clone SK1 (BD Biosciences), CD45-APC clone MB4-6D6 (Miltenyi), CD14-BV785 clone M5E2 (Biolegend), CD68-FITC clone Y1/B2A (eBioscience), and Live/Dead Aqua Fixable Dead Stain (Life Technologies) for 20 minutes at room temperature. After a wash in PBS + 2% FBS, cells were resuspended in PBS + 2% FBS and acquired on a BD LSRII flow cytometer with phagocytosed E. coli bioparticles detected on the PE filter. Data were analyzed using FlowJo software (version 1.1.0-SNAPSHOT).

Cryopreserved PBMC were stained for antigen-specific B cells and subsets (Figure S6) using the following panel: IgM BUV395 (clone G20-127, Becton Dickenson), CD8 BUV665 (clone RPAT8, BD), CD56 BUV737 (clone NCAM16, BD), IgD FITC (Southern Biotech), IgA Dy405 (polyclonal, Jackson Immunoresearch), aqua LIVE/DEAD (Invitrogen), CD14 BV785 (clone M5E2, BioLegend), CD20 Alexa700-PE (clone 2H7, vaccine research center), IgG Alexa700 (clone G18-145, BD), CD3 Cy7APC (clone SP34-2, BD Pharmingen). Biotinylated prefusion-stabilized spike (S-2P) and spike subdomain (NTD, RBD) probes were produced as previously described^{79 (link)} and conjugated to streptavidin-labeled dyes (BD) to yield the following and streptavidin-conjugated B cell probes, NTD SA-BB700 (BD), RBD SABV650 (BD), and S-2P SA-APC (BD). PBMC were thawed into cRPMI + 10% FBS^{85 (link)}, washed with PBS, and stained with aqua LIVE/DEAD kit in PBS for 20 min. at 4 °C. Staining was then completed with the remainder of the antibody and probe cocktail described above for 45 min. at 4 °C, and washed twice with PBS before flow cytometry.

Cytometry results represent at least three biological replicates for each time point on long-term culture and were obtained from a combination of seven experiments with mixes of 10 independent healthy donors. Cells were cultured in a prestimulation medium with or without metabolic drugs and αKG during the first 4 d and then in the optimized prestimulation medium without inhibitor or αKG as described before. A sample of cells were collected at days 4, 7, 10, and 14 to be analyzed by flow cytometry on CytoFLEX V5-B5-R3 Flow Cytometer (Beckman Coulter). After a saturation step with Gamma Immune (dilution 1:2; Sigma-Aldrich), cells were labeled with antibodies detecting the following cell surface markers: CD133-APC (clone 7, dilution 1:20; BioLegend), CD19-PE Cy7 (clone HIB19, dilution 1:80; BioLegend), CD36-PerCP-Cy5.5 (clone 5–271, dilution 1:40; BioLegend), CD45-BV421 (clone HI30, dilution 1:80; BioLegend), CD15-APC-Fire 750 (clone W6D3, dilution 1:80; BioLegend), CD14-BV785 (clone M5E2, dilution 1:80; BioLegend), CD41-BV605 (clone HIP8, dilution 1:40; BioLegend), and Zombie Aqua fixable viability dye (dilution 1:200; BioLegend). For each experiment, compensation beads (ref. 130-097-900; Miltenyi) were used for positive monolabeling controls and unmarked cells for negative controls.