Model 3550 uv

The MTT assay was employed to determine the number of viable cells in culture. Briefly, the cells were plated into 96-well plates and exposed to ethanol with/without C3G (20 μM) or PDTC (20 μM) for indicated times. After the treatment, 10 μl of MTT reagent was added into each well and the plates were incubated at 37°C for 4 h. After lightly vortexing the plate on an orbital shaker, the spectrophotometric absorbance was read on a microplate reader (Bio Rad Model 3550-UV) at 595 nm. Each individual experiment was performed at least three times.

HPLC activity-based profiling was realized using the post-chromatographic reaction of HPLC-eluates with the acetylcholinesterase/α-naphthyl acetate/Fast Blue B salt model system. Aliquots (100 µL) of extract solution (10 mg/mL) were separated under analytical HPLC conditions (Section 2.4). The eluates (50 µL) were collected every 30 s using an automated fraction collector (Econova) in 96-well plates, then dried under a N₂-steam and redissolved in 2.7 µL of DMSO. To each sample, 235.7 µL of 0.1 M phosphate buffer pH 7.4 and 20 µL of a solution acetylcholinesterase (final concentration 0.1 U/mL in 0.1 M phosphate buffer) and 1.6 µL of α-naphthyl acetate solution in DMSO were added. After mixing for 90 s and incubation at 25 °C for 90 s, 20 µL of a 5% solution of sodium dodecyl sulfate (SDS) in water and 20 µL of Fast Blue B salt solution in water (final concentration 0.17 mM) were added. The microplate was read by a Bio-Rad microplate reader Model 3550 UV (Bio-Rad Labs, Richmond, CA, USA). Enzyme activity and inhibition were quantified by determination of the absorbance at 600 nm after the formation of the purple-colored diazonium dye as a percentage. The percentage of inhibition was calculated relative to a control sample, for which the anti-acetylcholinesterase activity was assessed under identical conditions, but in the absence of the test compound.

Cell viability was assessed by using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma Aldrich, St. Louis, MO, USA) as previously described [61 (link)]. Cells were exposed to TTE (1–1000 μg/mL) for 24, 48, and 72 h. After treatments, MTT was added to wells and the plates were incubated at 37 °C for 4 h and the formazan crystals that formed were extracted with DMSO. The absorbance was read on a microplate reader (Bio Rad Model 3550-UV, Tokyo, Japan) at 595 nm. Experiments were performed at least three times. The concentration of extract required for 50% inhibition of the cell viability (IC₅₀) was determined by plotting the percentage of cell viability versus log concentration.

Before use, freshly collected human blood (O positive) was washed three times with 0.01 M Tris-HCl at pH 7.4, which contained 0.15 M NaCl (Tris-saline). A suspension of 1% (v/v) erythrocytes consisting of packed red blood cells resuspended in Tris-saline was prepared. A FLU-loaded ME, a FLU-unloaded ME, and a FLU-free sample were dissolved in Tris-saline to a final concentration of 27 μM. A 1% (v/v) Triton X-100 solution was used as a positive control (which corresponded to 100% lysis). After incubation for 1 h at 37°C, the samples were centrifuged at 3000 ×g for 2 min. Aliquots of 100 μL of the supernatant were transferred to 96-well microplates, and the absorbance was determined at 405 nm using a BioRad Model 3550-UV (USA) microplate reader. The assay was performed in triplicate. The percentage of haemolysis was calculated using the following equation: % haemolysis = (test sample absorbance/absorbance at 100% lysis) × 100 [38 (link), 39 (link)].

Treatment and collection of plasma samples as mentioned in the previous, plasma levels of CaSR, cAMP, renin and Ang II (Ang II)were determined by using standard commercially available enzyme immunoassay for the in vitro quantitative measurement in rats and human (USCN Life Science, Wuhan, China), intra-assay and inter-assay coefficients of variation less than 8%. The absorbance at 450 nm was read using a Microplate Reader (Bio-RAD Model 3550-UV, USA).

The incorporation of tritiated [methyl-³H]-thymidine into primary astrocytes was conducted according to established procedures to quantify cell proliferation^{23 (link)–25 (link),59 (link),61 (link)}. Radio-labeled [methyl-³H]-thymidine (25 Ci/mmol; Amersham, Arlington Heights, IL) was added to each well 1 h prior to the termination of the experiment at a final activity of 1.0 μCi/ml (37 °C, 5% CO₂/95% humidified air). At the end of the incubation, cultures were washed twice with 2 mL of Tris-EDTA buffer (pH 7.4) to remove excess ³H-thymidine. Then, DNA and total cellular proteins were extracted using the trichloroacetic acid (TCA) precipitation method^{59 (link),61 (link)}. Cell proliferation was measured as the incorporation of radioactivity per μg of protein in the acid-precipitated portion (cpm/μg protein). Tritium was quantified with a scintillation counter (LKB Wallac, Gaithersburg, MD) for beta particles for 10 min using an Ecoscint-A liquid scintillation cocktail (National Diagnostics, Manville, NJ). Total cellular protein was determined by the Bradford assay (Bio-Rad, Hercules, CA) using a microplate reader (model 3550-UV, Bio-Rad) at a wavelength of 595 nm.