Cell apoptosis was detected using flow cytometric assay with Annexin V\FITC and PI\staining. of diabetes mellitus (DM). In this study, we evaluated the effect of lentinan (LNT), an active ingredient purified from the bodies of and has been used in traditional medicine 16, Tamsulosin hydrochloride 17. Many previous studies have demonstrated that LNT exhibited multiple bioactivities, including antioxidation 18, antitumour 19, antiviral 20, antibacterial 21, 22, anti\inflammation 23 and immunoregulation 24. However, LNT has not been used for the treatment of diabetes, and an effect of LNT on cells has not been reported. Therefore, in this study we designed experiments to investigate whether LNT can protect against pancreatic \cell apoptosis and dysfunction induced by streptozotocin (STZ). Furthermore, we investigate the mechanisms underlying of this protective action, to determine whether it might be a potential pharmacological treatment of stress\mediated diabetes. Materials and methods Cell culture A rat INS\1 cell line, purchased from American Type Culture Collection (ATCC, Manassas, VA, USA), retains physiological characteristics of normal cells. INS\1 cells (passages 10C20) were grown in RPMI 1640 medium (Hyclone, Logan, UT, USA), containing 6% fetal bovine serum (FBS) (vol./vol.), 50 mol/l \mercaptoethanol, 1 mmol/l sodium pyruvate, 2 mmol/l L\glutamine, 100 U/ml penicillin, 100 g/ml streptomycin (all from Sigma\Aldrich, St. Louis, MO, USA) and cultured at 37C in a humidified atmosphere containing 95% air and 5% CO2. Cell viability assay Cell viability was determined by an MTT [3\(4,5\dimethyl\2\thiazolyl)\2,5\diphenyl\2\H\tetrazolium bromide] assay. Briefly, INS\1 cells were seeded in 96\well plates at a density of 1 1 104 cells per well. Some cells were treated with STZ at Tamsulosin hydrochloride concentrations of 0, 0.25, 0.5, 1 and 2 mmol/l for 24 hrs, followed by incubation with MTT (0.5 mg/ml, Sigma\Aldrich) for 4 hrs. Other cells were treated with LNT (Sigma\Aldrich), which was dissolved in physiological saline. Following pre\incubation with LNT at concentrations of 0, 50, 100, 200 and 400 g/ml for 30 min., the cells were exposed to STZ (0.5 mmol/l) and LNT (0, 50, 100, 200 and 400 g/ml) for an additional 24 hrs. Each well was then supplemented with 10 l MTT and incubated for 4 hrs at 37C. Then, the formazan precipitate was dissolved in dimethyl\sulfoxide (Sigma\Aldrich) and the absorbance at 490 or 570 nm was determined Tamsulosin hydrochloride with a microplate reader (Perlong, Beijing, China). EdU proliferation assay Cell proliferation was measured by 5\ethynyl\2\deoxyuridine (EdU) assay using an EdU assay kit (Ribobio, Guangzhou, China) according to the manufacturer’s instructions. Briefly, INS\1 cells were seeded at 2 103 cells per well in 96\well plates and pre\incubated with indicated LNT (50, 100, 200 and 400 g/ml) in a humidified atmosphere containing 5% CO2 at 37C for 30 min. After 30 min. of incubation, the cells were treated with STZ (0.5 mM) and the indicated concentration of LNT and further incubated for 24 hrs. Then, the cells were incubated with 50 M EdU for additional 3C4 hrs at 37C before fixation and permeabilization. After 3 washes with PBS, the cell nuclei were stained with 100 l of Hoechst 33342 (1 g/ml) for 5C10 min. and visualized under a fluorescent microscope (Olympus, Tokyo, Japan). TUNEL staining assay INS\1 cells were cultured on coverglass in 12\well plates. After 24 hrs treatment as described above, the apoptotic cells were stained in a terminal deoxynucleotidyl transferase mediated nick\end labelling (TUNEL) assay according to the instruction of the kit manufacturer (In Situ Cell Death Detection Kit; Roche, Basel, Switzerland) 25. Apoptotic cells were stained by green fluorescence, and all cells were marked with blue fluorescence using Hoechst. The apoptotic ratio was calculated as tunnel\positive cells divided by total cell number. The number of cells was counted in five random fields from three different slides at 400 magnification. An average for the percentage of tunnel\positive cells was taken over these fields. Flow cytometry analysis INS\1 cells (1 106 cells per well) were cultured in 6\well plates and pre\treated with LNT or anisomycin (Am; Sigma\Aldrich), ENAH a direct activator of JNK and p38, for 30 min. and then exposed to STZ or LNT an additional 24 hrs. Thereafter, the cells Tamsulosin hydrochloride were digested with 0.25% trypsin and incubated with 20 l of binding buffer, 5 l of Annexin V\FITC and 5 l of propidium iodide. After incubation at room temperature in the dark for 15 min., cells were analysed by flow cytometry 26. The results were calculated using the CellQuest? Pro software (BD Biosciences, San Jose, CA, USA) and expressed as the percentage of apoptotic cells from the total cells. Measurement of the intracellular reactive oxygen species generation A commercial.