Bacterial lipopolysaccharide

Xuebijing injection (catalog number: z20040033, batch number: 1905061) was manufactured by Tianjin Chase Sun Pharmaceutical Co., Ltd (Tianjin, China). Paeoniflorin (CAS #:23180-57-6), Hydroxysafflor yellow A (CAS #: 78281-02-4), Ferulic acid (CAS # 537- 98-4), and protocatechuic aldehyde (CAS #: 139-85-5) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Mouse NT-proBNP ELISA (Cat#: ZC-37812-96T) kit, DCFH-DA (Cat#:CA1410-100T), RIPA tissue/cell lysate (Cat#: R0020), SDS-PAGE gel kit, and Rainbow 245 Spectrum Marker (Cat#:PR 1920), Mouse IL-6 ELISA kit (Cat#:ZC-37988-96T), Mouse IL-1β ELISA kit (Cat#:ZC-37974-96T), and EasySee Western Blot Kit (Lot#:O30807) were purchased from Solarbio (Beijing, China). The primary antibody of rabbit anti-CXCL2 (bs-1162R) was purchased from Bioss Inc., (Beijing, China) and GAPDH, 1:4,000 (14C10) was from Cell Signaling Technology (Beverly, MA, United States). The secondary antibody Goat anti-Rabbit IgG (1:4,000) (ZB-2301) was purchased from ZS bio (Beijing, China). 2, 2, 2-Tribromoalcohol (Cat#:T48402) and Bacterial lipopolysaccharide (Cat#: L2880-10 MG) were purchased from Sigma Aldrich (St. Louis, MO, United States). Rhod2-AM (Cat#: zy0129) and Anti-GRP78 (BiP) antibody (ab21685) were purchased from Abcam (Cambridge, MA, United States).

Injections were performed using a tabulated microcapillary pipette (Sigma-Aldrich). Saline solution was used as vehicle (5 mM KCl, 10 mM NaH₂PO₄, pH 7.8) as previously described (Mustard et al., 2010 (link)). Bacterial lipopolysaccharide (LPS; Escherichia coli LPS, serotype 0111:B4, Sigma-Aldrich, St. Louis, USA) was diluted in vehicle at 1 mg/ml, aliquoted and conserved at −20°C until used. In all cases, 4 μl of saline or LPS were injected into the thorax of the bees (Felsenberg et al., 2011 (link)).

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT), Griess reagent and bacterial lipopolysaccharide (LPS) were acquired from Sigma Chemical Co. (St Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Life Technologies (Auckland, New Zealand). Dulbecco’s Modified Eagle’s Medium (DMEM), Penicillin-streptomycin solution, Dulbecco’s Phosphate Buffered Saline (DPBS), GlutaMAX™-1 and 0.25% Trypsin-EDTA were obtained from Hyclone (Logan, Utah, USA). PE-conjugated rat anti-mouse TLR4/MD-2 complex (clone MTS510) and rat IgG2a kappa isotype control antibodys were products of eBioscience (San Diego, CA, USA). Mouse TNF-α and IL-2 ELISA kits were acquried from Neobioscience Technology Co., Ltd. (Beijing, China). NF-κB Activation –Nuclear Translocation Assay kit from Beyotime Institute of Biotechnology (Nanjing, China). Rabbit phospho-NF-κB (Ser536) antibody and rabbit NF-κB p65 (C22B4) antibody from Cell Signaling Technology (Danvers, MA) were also obtained. Epimedin C and icariin were provided by the National Institute for Food and Drug Control (Beijing, China). Epimedin B was purchased from Herbest (Baoji, China). Baohuoside II and Baohuoside VII were obtained from Chem Faces (Wuhan, China). All standards had a purity of 98%.