Lipopolysaccharide lps from escherichia coli

CL429 (Cat. Code: vac‐c429) was purchased from InvivoGen. Lipopolysaccharide (LPS) from Escherichia coli was purchased from Sigma (St.Louis, MO, USA), and Pam3CSK4 was obtained from EMC Microcollection (Tübingen, Germany). The TransDetect Annexin V‐FITC/PI Cell Apoptosis Detection Kit was purchased from TransGen Biotech (Beijing, China). RPMI 1640 and foetal bovine serum (FBS) were supplied by Gibco. Streptavidin FITC, anti–Mouse CD117 APC eFluor 780, and anti‐Mouse Ly‐6A/E (Sca‐1) PerCP‐Cyanine5.5 were purchased from BD Pharmingen (San Diego, CA). Lineage Cell Detection Cocktail‐Biotin was purchased from Miltenyi Biotec (Germany). Lipofectamine 3000 was purchased from Thermo Fisher. Antibodies were purchased from Affinity Biosciences (Jiangsu, China) and Cell Signaling Technology (Massachusetts, USA). Commercial ELISA kits were purchased from DAKEWE (Beijing, China).

The EA was collected in Asan, South Korea, in November 2020, and the plants were air-dried and stored at −20°C before use. Two types of ethanol extracts, EA leaf extracts 1 and 2, were prepared. The dried leaves of EA (2 kg) were extracted with 70% ethanol (2 × 20 L) for 4 h (two times) at 80°C. The extract was filtered through a filter paper. The solvent was evaporated under reduced pressure to yield the concentrated leaf extract 1 (174.0 g). Next, leaf ethanol extract 1 (9.0 g) was suspended in 70% ethanol and fractionated with hexane (four times). A portion of 70% ethanol was evaporated under reduced pressure to yield the concentrated leaf extract 2 (6.7 g). Chlorophyll-free leaf extract 2 was used for in vitro and in vivo studies. Lipopolysaccharide (LPS) from Escherichia coli and Cy or HemoHIM were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Kolmar BNH Co., Ltd. (Sejong, Korea), respectively.

Tissue culture media and media supplements, mannan (Saccharomyces cerevisiae), laminarin (Laminaria digitata), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), f-MLF (N-Formyl-methionyl-leucyl-phenylalanine), epicatechin, polyinosinic:polycytidylic acid, sodium salt (poly (I:C)), lipopolysaccharide (LPS) from Escherichia coli, TNF-alpha, and GM-CSF were obtained from Sigma-Aldrich (St. Louis, USA). 4-(N-Maleimidomethyl) cyclo-hexanecarboxylic-acid N-hydroxysuccinimide ester (SMCC) was provided by Thermo Scientific (Erembodegem, Belgium). Biocompatible Anchor for cell Membrane (BAM, Mw 4000) was obtained from NOF EUROPE (Grobbendonk, Belgium). N-Formyl-methionyl-leucyl-phenylalanine with two lysine molecules (f-MLFKK) was synthesized by Schafer-N (Copenhagen, Denmark). Imiquimod (R-837) was delivered by Merck Millipore (Billerica, USA), monophosphoryl lipid A (MPLA) by Avanti Polar Lipids (Alabaster, USA), and resiquimod (R-848) by Tocris Bioscience (Bristol, UK).

C57BL6 BM-derived immature DCs (imDCs) were generated as described previously [16 (link), 17 (link), 29 (link)]. Briefly, BM was harvested from the femurs and tibiae of mice and cultured in RPMI-1640 medium (Gibco-BRL) supplemented with 10% (v/v) FBS (Gibco-BRL) and 1% (w/v) PS in the presence of 20 ng/mL recombinant murine (rm) GM-CSF (R&D Systems, Minneapolis, MN, USA) and 10 ng/mL rmIL-4 (R&D Systems). On culture days 2 and 4, half of the medium was removed and replaced with fresh media containing cytokines. On day 6, imDCs were purified via positive selection with CD11c⁺-magnetic beads (Miltenyi Biotec Inc., Auburn, CA, USA). Then, mature DCs (mDCs) were generated by further cultivation for 48 h of CD11c⁺ DCs with 1 μg/mL lipopolysaccharide (LPS) from Escherichia coli (Sigma-Aldrich) and 10 ng/mL rmGM-CSF.