BMMCs were cultured for 5 days under continuous high-dose 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) stimulation or PBS control conditions. For trehalose 6,6’-dimycolate (TDM; invivoGen) experiments, cells were maintained in TDM (10 µg/mL) for the full 5 day LPS exhaustion time course. For in vivo experiments, sepsis was induced by intraperitoneal injection of cecal slurry in 6- to 8-week-old C57BL/6 WT male mice as previously described. Briefly, whole ceca were dissected from 12-week-old C57BL/6 mice euthanized by cervical dislocation. Cecal contents were extracted and mixed with sterile water (250 µg/µL), sequentially filtered through 860 µm and 190 µm mesh strainers, mixed with an equal volume of 30% glycerol in PBS (final concentration 125 µg/µL), and placed in -80˚C storage. For cecal slurry injections, frozen cecal slurry (CS) stock was rapidly thawed in 37˚C water bath and injected (0.9 mg/g mouse weight) into the peritoneal cavity of recipient mice. After 5 days, mice were injected with a second 0.9 mg/g dose of CS, and then euthanized by cervical dislocation 1, 2, or 7 days later. Bone marrow cells were collected as previously described and used for flow cytometry analysis or LS column purification (Miltenyi) of bone marrow monocytes following the manufacturer’s protocol.
Growth protocol
In vitro bone marrow monocyte (BMMC) LPS exhaustion experiments were performed as previously described (Pradhan et al., 2021, Front. Immunol.). Briefly, bone marrow cells were harvested from 6- to 8-week-old C57BL/6 WT female mice, seeded at a density of 3x105 cells/cm2, and cultured in complete RPMI 1640 media (10% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamine) supplemented with 10 ng/mL M-CSF (PeproTech). Cells were cultured for 5 days at 37˚C in a humidified 5% CO2 atmosphere under continuous high-dose 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich) stimulation or PBS control conditions, with fresh media changes at days 2 and 4 of culture. For trehalose 6,6’-dimycolate (TDM; invivoGen) experiments, cells were maintained in TDM (10 µg/mL) for the full 5 day LPS exhaustion time course.
Extracted molecule
genomic DNA
Extraction protocol
Cultured BMMCs were harvested by manual disruption and either directly processed for genomic DNA extraction (total monocytes) or FACS sorted based on Ly6C expression (Ly6C-low, -int, and -high). Bone marrow monocytes from in vivo experiments were isolated using LS columns (Miltenyi). Genomic DNA was prepared for all samples using a DNeasy Blood and Tissue Kit (Qiagen).
