Raw264.7 cell line

Twelve batches of Ganoderma lucidum (numbered S1 to S12) fruit bodies were collected from different places in China, including S1~S9 from Shouxiangu, Zhejiang Province, S10 from Huangshan, Anhui Province, and S11~S12 from Longquan, Zhejiang Province. The sample information is listed in Table 1. Trifluoroacetic acid (TFA), 1-phenyl-3-methyl-5-pyrazolone (PMP), monosaccharide standards (rhamnose, glucosamine, glucose, mannose, galactose, fucose, arabinose, fructose, xylose, glucuronic acid, and galacturonic acid), lipopolysaccharide (LPS), and polymyxin B were purchased from Sigma-Aldrich (St. Louis, MO, USA). Oligosaccharide standards including laminaribiose, laminaritriose, laminaritetraose, laminaripentaose, and laminarihexaose were purchased from Megazyme (Wicklow, Ireland). Pullulan standards P-5 (M_w = 6300 g/mol) and P-10 (M_w = 9800 g/mol) were purchased from Shodex (Tokyo, Japan). The RAW264.7 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences. DMEM medium and fetal calf serum were bought from Gibco (Grand Island, NY, USA). The mouse TNF-α ELISA kit was bought from Beijing 4A Biotech Co., Ltd. (Beijing, China). All other reagents were analytical grade and produced in China.

RAW264.7 cell line was acquired from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, United States) added with 10% fetal bovine serum (FBS, Biological Industries, Israel), and cultured at 37°C containing 5% CO₂ in an incubator. RAW264.7 cells grown in DMEM medium were stimulated by lipopolysaccharide (LPS, 1 µg/ml, L2880, Sigma-Aldrich, St. Louis, MO, United States) for 24 h to polarize M1 macrophages, while M2 macrophages were induced by IL-4 (15 ng/ml, CK74, novoprotein, shanghai, China) treatment for 24 h.

Escherichia coli strains were incubated at 37 °C in Luria–Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) with 50 mg/L kanamycin. Authentic G-Rb1 and Gyp XVII were purchased from Shanghai Yuanye Biological Technology Co. Ltd. (Shanghai, China). The p-nitrophenyl β-d-glucopyranoside (pNPβGlu), p-nitrophenyl α-d-glucopyranoside (pNPαGlu), pNP-α-l-arabinofuranoside (pNPαAraf), pNP-α-l-arabinopyranoside (pNPαArap), p-nitrophenyl β-d-lactose (pNPβLac), p-nitrophenyl β-d-galactose (pNPβGal), p-nitrophenyl β-d-mannose (pNPβMan), p-nitrophenyl α-l-mannose (pNPαMan), p-nitrophenyl β-d-xylose (pNPβXyl), p-nitrophenyl β-d-fucose (pNPβFuc), p-nitrophenyl β-d-cellobioside (pNPβCel), pNP-α-l-rhamnopyranoside (pNPαRha), and carboxymethyl cellulose (CMC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, methanol, and formic acid of HPLC grade were obtained from Fisher Scientific (Waltham, MA, USA).
The RAW264.7 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Gibco (Carlsbad, CA, USA). Lipopolysaccharides (LPS) were purchased from Sigma (St. Louis, MO, USA). A Cell Counting Kit-8 (CCK-8) and enzyme-linked immunosorbent assay (ELISA) kit were obtained from Bestbio (Shanghai, China) and R&D (Minneapolis, MN, USA).

Escherichia coli strains were incubated at 37 °C in Luria–Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, and 10 g/L NaCl) with 50 mg/L kanamycin. Authentic G-Rb1 and Gyp XVII were purchased from Shanghai Yuanye Biological Technology Co. Ltd. (Shanghai, China). The p-nitrophenyl β-d-glucopyranoside (pNPβGlu), p-nitrophenyl α-d-glucopyranoside (pNPαGlu), pNP-α-l-arabinofuranoside (pNPαAraf), pNP-α-l-arabinopyranoside (pNPαArap), p-nitrophenyl β-d-lactose (pNPβLac), p-nitrophenyl β-d-galactose (pNPβGal), p-nitrophenyl β-d-mannose (pNPβMan), p-nitrophenyl α-l-mannose (pNPαMan), p-nitrophenyl β-d-xylose (pNPβXyl), p-nitrophenyl β-d-fucose (pNPβFuc), p-nitrophenyl β-d-cellobioside (pNPβCel), pNP-α-l-rhamnopyranoside (pNPαRha), and carboxymethyl cellulose (CMC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile, methanol, and formic acid of HPLC grade were obtained from Fisher Scientific (Waltham, MA, USA).
The RAW264.7 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Gibco (Carlsbad, CA, USA). Lipopolysaccharides (LPS) were purchased from Sigma (St. Louis, MO, USA). A Cell Counting Kit-8 (CCK-8) and enzyme-linked immunosorbent assay (ELISA) kit were obtained from Bestbio (Shanghai, China) and R&D (Minneapolis, MN, USA).

Ginseng root extract was purchased from ZeLang Healthtech Co. Ltd., China. Reference ginsenosides, including ginsenoside Rb1, Re, Rg1, Rd, Gyp XVII, Rg2, F1, Rh1 and PPT were purchased from Jilin University (Changchun, China). Acetonitrile and methanol of HPLC grade were obtained from Fisher Scientific (Waltham, MA). HPLC grade formic acid (96%) was obtained from Tedia (Fairfield, OH). Other reagents were of analytical purity.
The RAW264.7 cell line was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Gibco (Carlsbad, USA). Lipopolysaccharides (LPS) was purchased from Sigma (Missouri, USA). A Cell Counting Kit-8 (CCK-8) and enzyme-linked immunosorbent assay (ELISA) kit were obtained from Bestbio (Shanghai, China) and R&D (Minneapolis, USA).

The RAW264.7 cell line was bought from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). They were cultured in DMEM medium containing 10% FBS, 1% Penicillin/Streptomycin and kept in a humidified air with 5% CO₂ at 37 °C. When the cells grow at a fusion degree of about 70–80%, they are subcultured. Straight after, collect the cells with cells gently in a centrifuge tube to centrifuge (800 rpm, 5 min). Subsequently, discard the supernatant and add 1 mL medium, blow up the cells with a pipettor. Finally, put the cells in culture flask and place them in the incubator. Collect the cell’s pellets in the logarithmic growth phase, and adjust the cell density according to the requirements of the number of cells in the seed plate before further treatments.