Lps from e coli 026 b6

The RAW 264.7 macrophage cell line was obtained from Sigma-Aldrich (USA) and grown to confluence in DMEM containing l-glutamine (2 mM), penicillin (100 IU/mL), streptomycin (100 mg/mL), and 10% FBS at 37 °C in a 5% CO₂ humidified incubator. Macrophages were plated in six-well plates at a concentration of 3 × 10⁵ cells in 2 mL per well. After this, MSCs were also co-cultured in a proportion 1:10 with the macrophages in the same well. After 24 h, macrophages and macrophages co-cultured with MSCs were stimulated with LPS from E. coli 026:B6 (1 μg/mL; Sigma-Aldrich) at different times.

To test the impact of different forms of β-Glucans on mice, the glucans were administered with and without LPS in mice. Mice were intraperitoneally (ip) administered with 10 mg/kg of LPS from E. coli 026: B6 (Sigma-Aldrich) with or without intravenous (iv) administration (tail vein injection) of the different glucans at 20 mg/kg. Blood samples were collected through the tail vein at several timepoints, and mice were sacrificed at 24 h post-injection by cardiac puncture under isoflurane anesthesia before sample collection.

LPS from E. coli 026:B6 (Sigma-Aldrich) and ATP (Invivogen) were used at concentration of 50 ng/ml and 3 mM for human and 100 ng/ml and 5 mM for murine co-culture studies, respectively. Nigericin (Invivogen) was used at a concentration of 10 µM. To evaluate autophagy response, WT and WAS KO BMDCs or WT and WAS KO THP-1 cells were stimulated with rapamycin (50 nM; Calbiochem) and bafilomycin (160 nM; Thermo Fisher Scientific) alone or in combination. All murine IFNs were from Millipore and human cytokines from Peprotech. BMDCs were stimulated with 500 U/ml IFN for 16 h prior to LPS priming (100 ng/ml) for 3 h, followed by ATP (5 mM) for 30 min.

Mice were administered an i.p. injection of 35 µg LPS from E. coli 026:B6 (Sigma Aldrich) diluted into 500 µL saline. This quantity of LPS is in the range of the protective amount [see Ref. (37 (link))]. After 8 h, submandibular blood and tissues were collected from each animal. Blood was collected using Goldenrod lancets (Medipoint, Inc.) in K₂ EDTA microtainers (BD Biosciences) and centrifuged at 10,000 × g for 5 min. Plasma was removed, aliquoted, and stored at −80°C until use. ELISAs for IL-18 (eBioscience) and IFN-γ (BioLegend) were performed according to manufacturers’ recommendations.

Human leukemia monocytic cells of the THP-1 cell line (TIB-202 ^TM; ATCC, Manassas, VA, USA) were cultured in RPMI 1640 (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), 100 U/mL penicillin, 100 µg/mL streptomycin (Invitrogen), and 50 µM 2-mercaptoethanol. After that, THP-1 cells were incubated with 100 ng/mL of phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) for macrophage differentiation [30 (link)]. Then, the differentiated THP-1 cells were stimulated with RPMI (untreated), LPS from E. coli 026:B6 (100 ng/mL) (Sigma-Aldrich), interleukin 4 (IL-4) (20 ng/mL), and 20% tumor-conditioned media prepared by centrifuging 5 × 10⁶ CaCO2 and HepG2 cells (cancer cell lines) incubated in the presence or absence of BAM15 (50 ng/mL) [31 (link)]. At 24 h after stimulation, supernatants were collected for cytokine quantification using ELISA (Invitrogen), and the cells were harvested for quantitative real-time polymerase chain reaction (PCR).