Before being added such to cells, the DCQD stock solution was diluted with PBS to prepare the working solutions. The final DMSO concentrations were all less than 0.1% when DCQD was added to cells. The dose of DCQD was calculated and diluted according to its contents quantitatively analyzed by HPLC system. Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). DMSO, Cerulein, F12K medium and DCFH-DA were obtained from Sigma (St. Louis, MO, USA). 1.2. Rats Sprague-Dawley rats (243��18 g) were purchased from the Experimental Animal Center of West China Center of Medical Sciences of Sichuan University. All animal studies were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
The protocol was approved by the Committee on the Ethics of Animal Experiments of the Sichuan University. 2. Methods 2.1. Cell culture Rat pancreatic acinar AR42J cells (ATCC, Rockville, MD, USA) were cultured in F12K medium containing 20% FBS and 100 U/mL penicillin, 100 ��g/mL streptomycin in standard condition (37��C and 5%CO2). All experiments were carried out 24 h after cells were seeded. To investigate the protective effects of DCQD against AP, AR42J cells were treated with or without DCQD prior for 30 min, then further co-incubated with cerulein (10?8 M) for another 24 h. 2.2. Cell viability assay Cell survival was assessed by WST viability assay kit containing WST-8(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) according to the manufacturer’s protocol (Roche, Basel, Switzerland).
AR42J cells were plated in 96-well plates at 2��104 cells/well. After 24 h incubation, cells were pretreated with or without DCQD at different concentrations (0�C0.004 g/mL) and then were further co-incubated with cerulein for 24 h, WST-8 solution (0.5 mg/mL) was added to each well and incubated at 5% CO2 37��C for 2 h. The cell viability was determined by the differences absorbance at wavelengths of 450 nm and 630 nm. The relative cell viability rate was calculated according to the following formula: Cell viability rate (%)=100%��mean absorbance of cells in sample groups/mean absorbance for cells in control group. 2.3. LDH assay Necrotic cell death was assessed by the release of lactate dehydrogenase (LDH) from the cytosol of the damaged cells into the supernatant [15], using the LDH cytotoxicity detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) for various time point 0�C24 h according to the manufacturer’s instructions. Values for LDH release are presented as the percentage of total cellular LDH from the following equation [16]: LDH release Anacetrapib (%)=total extracellular LDH activity at the time point��100/total LDH activity. 2.4.