A database was prepared for the identification of phytoconstituents of http://www.selleckchem.com/products/AG-014699.html HLJDT based upon their molecular masses, calculated m/z values and retention times (Rt) observed in the analysis (Table S1). Representative LC-MS base peak chromatograms (BPC) and extracted ion chromatograms (EIC) are shown in Figure S1. By matching the Rt and m/z values between samples and reference standard solutions (Table S2), eight characteristic peaks of HLJDT were identified in HLJDT sample solutions as geniposide, coptisine, jatrorrhizine, baicalin, palmatine, berberine, baicalein, wogonin, phellodendrine, columbamine, epiberberine and wogonoside were identified based on published articles related to the profiling of chemical components of HLJDT [21],[22]. Cell Culture N2a-SwedAPP cells [23] were obtained from Dr.

Gopal Thinakaran (University of Chicago, Chicago, IL, USA). N2a-SwedAPP cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and OPTI-MEM medium in a 11 ratio with 5% FBS, 50 ��g/mL penicillin, 50 ��g/mL streptomycin and 200 ��g/mL G418 as previously described [23]. N2a-SwedAPP cells were incubated at 37��C in a 5% CO2/95% humidity incubator. Cells were seeded 24 h prior to the treatments. When growth reached close to 90% of confluence, cells were transferred into DMEM with 1% FBS for loading extracts. The final concentration of DMSO in all experiments was 0.1%, and this concentration caused no cytotoxicity. Viability Assay For the viability assay, N2a-SwedAPP cells were seeded in a 96-well plate (7000 cells/well and 5000 cells/well) for 48 h.

The exhausted medium was replaced after 24 h with 200 ��L DMEM with 1% FBS and various concentrations (0, 3.125, 6.25, 12.5, 25 or 50 ��g/mL) of each extract or various concentrations (0, 3.125, 6.25, 12.5 or 25 ��M) of berberine, baicalein or geniposide, and cells were incubated at 37��C in 5% CO2 for 48 h. Then the media were removed and 100 ��L of phenol red-free DMEM containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added to each well to a final concentration of 0.5 mg/mL and further incubated for 4 h. The MTT-containing medium was removed, and the cell crystals were dissolved using 100 ��L of 20% sodium dodecyl sulfate (SDS) in 50% N, N-dimethylformamide. Finally, optical intensity was measured using a BioRad plate reader at 570 nm and a reference of 620 nm. Experiments were conducted independently three times. Detection of Intracellular APP and Soluble APPs in N2a-SwedAPP Cells To detect the levels Anacetrapib of APP metabolic products, the cell lysates and the conditioned media were prepared as described previously by Durairajan et al. [24], with minor modification.