Kz 3 f

Intestinal tissues and peripheral blood from aforementioned rats in the qRT-PCR experiment were harvested for detection of inflammatory responses and intestinal barrier permeability using ELISA. Tissues were ground using a homogenizer (KZ-III-F, Servicebio, Wuhan, China) and reconstituted in PBS. The samples were centrifuged at 12,000 × g and 4 °C for 10 min, and the supernatant was collected for ELISA. Peripheral blood was centrifuged at 2000 × g and 4 °C for 10 min to prepare plasma samples. ELISA kits for rat tumor necrosis factor-α (TNF-α, Abbkine, Wuhan, China), interleukin-6 (IL-6, Abbkine), diamine oxidase (DAO, Enzyme-linked Biotechnology, Shanghai, China), D-lactate (Nanjing Jiancheng, Jiangsu, China) and endotoxin (Enzyme-linked Biotechnology) were used to detect the levels of the corresponding indicators according to the manufacturer’s protocols. The absorbances were measured with a microplate reader (Biotek, VT, USA). The linear standard curves were determined using standards provided by the kits. The actual concentrations of these indicators were calculated based on standard curves.

Whole lung tissue, which BALF was not acquired from, was removed surgically and placed into 2 mL tubes which contained 3 3 mm metal beads with 1 mL of PBS and stored on ice. Tubes were placed in the KZ-III-F (Servicebio, 70 Hz, homogenization time: 70 sec). Lung homogenate were plated on Pseudomonas isolation agar plates and grown at 37°C overnight. Colonies were counted, and the total CFU/mL recovered from the homogenized tissue samples was calculated.

Four different species of cone snails (C. imperialis, C. litteratus, C. virgo, and C. marmoreus) were collected in the South Sea of China. The mitochondrial genome of cone snails was extracted by an improved high-salt precipitation method. In brief, approximately 0.15 g of muscle tissue of the gastrofoot was weighed, and 1ml of Buffer I (2 mmol/L EDTA, 5 mmol/L Tris, 7 mmol/L sucrose, and 220 mmol/L dulcitol) was added to it, and the pH was adjusted to 7. Next, the tissue was homogenized in an ice-cold tissue grinder (ServiceBio, KZ-III-F, Wuhan, China) then centrifuged at 800× g for 10 min at 4 °C, and the supernatant was collected. This procedure was repeated twice, then the supernatant was centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was discarded, and the precipitation was the isolated mitochondria. Then, 400 µL of Buffer Ⅱ (100 mmol/L Tris, 40 mmol/L Nacl, and 2 mmol/L EDTA), 16 µL of 20% SDS, and 16 µL of proteinase K (20 mg/mL) were added, after which the mitochondria was resuspended and incubated in a 60 °C water bath for at least 8 h until the solution was clear. Next, 300 of µL NaCl solution (40 mmol/L) was added and centrifuged at 8000× g for 10 min at 4 °C. The precipitation was discarded, and supernatant was collected. Then, isopropanol was added and centrifuged at 12,000× g for 15 min at 4 °C to precipitate the isolated mitochondrial DNA.