Dri chem nx500i

To prove that ligation of hepatic arteries does not have any detrimental effect on animal health, rat blood was collected from the tail vein 7 d prior to, at day of (4 h after), and then 2, 7 and 28 d after the surgery in experimental groups A, B, C and D. The serum was prepared and examined for following biochemical parameters: alkaline phosphatase (ALP), gamma-glutamyltransferase (GGT), aspartate transaminase (AST) and alanine transaminase (ALT), total bilirubin (TBIL), and total protein (TP) level, using DRI-CHEM NX500i automated clinical chemistry analyzer (Fujifilm, Japan).

Collected blood was centrifuged for 15 min at 13,000 rpm at 4℃. Total plasma
calcium and phosphorus in normal and malformed fish were measured respectively
using biochemistry automatic analyzer (FUJI DRI-CHEM NX 500i, Fujifilm Co.,
Japan). Plasma estradiol-17β (E2) level was measured by radioimmunoassay (RIA)
according to the Kobayashi’s method (Kobayashi
& Mikuni, 1987). Antiserum for E2 was purchased from Cosmo-Bio
Co., Ltd. (Tokyo, Japan). Radio-labeled steroids ([2,4,6,7-³H]-E2)
was purchased from Amersham Lifesciences (England). For the assay, steroids from
plasma were extracted twice in 2 mL diethyl ether, dried under nitrogen gas and
were dissolved in 0.1% gel-PBS.

A total of 24 male BALB/c mice were randomly divided into four groups (6 mice per group): vehicle alone, bletinib alone, LPS control, and bletinib treatment (bletinib + LPS). The mice were starved overnight and then injected i.p. with 50 μl of bletinib (25 mg·kg⁻¹) or 50 μl of vehicle (10% DMSO). ALI was induced through intratracheal spraying of 50 μl of LPS (from Escherichia coli O111:B4; 2 mg·kg⁻¹) or 50 μl of 0.9% saline (in vehicle and bletinib alone group) under general anaesthesia with xylazine (6 mg·kg⁻¹) and Zoletil 50 (tiletamine and zolazepam, 1:1; 30 mg·kg⁻¹). Five hours later, mice were killed (CO₂ asphyxia), the lungs removed and frozen at −80°C or fixed with 10% formalin. The serum levels of glutamic‐pyruvic transaminase (GPT), glutamic‐oxaloacetic transaminase (GOT), creatinine (CRE), and blood urea nitrogen (BUN) were determined by automated clinical chemistry analyser (Dri‐Chem NX500i, Fujifilm®, Japan). The level of blood neutrophils was measured using an automated haematology analyser (XT‐1800i, Sysmex Corporation, Kobe, Japan).
For the LPS‐induced mortality model, mice were injected i.p. with a single 50‐μl dose of LPS (from Escherichia coli O111:B4; 5 mg·kg⁻¹) or 0.9% saline (vehicle alone group). The mice were monitored for 5 days to determine survival.