Clariostar multiplate reader

Cell growth was measured with the In vitro Toxicology Assay kit (Sulforhodamine B, Sigma Aldrich, Saint Quentin Fallavier, France) according to manufacturer’s instructions. Cells were plated at a density of 2000 cells per well in 96-well plates in triplicates. Absorbance was measured at 565 nm using the CLARIOstar multiplate reader (BMG Labtech, Champigny-sur-Marne, France) at indicated time points.

ELISAs for quantifying total and serine-9 phosphorylated GSK3β (CST7265C PathScan® Total GSK3β Sandwich ELISA; CST7311C PathScan® Phospho-GSK3β (Ser9) sandwich ELISA) were obtained from Cell Signaling Technology. UDCA and vehicle (DMSO) treated neurons were processed according to the manufacturer’s instructions. Briefly, neurons were washed with PBS and lysed in kit cell lysis buffer (CST9803) containing 1 mM phenylmethylsulfonyl fluoride, protease and phosphatase inhibitors (Complete, Roche). Protein concentrations were determined using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) and an equal amount of total protein (0.15 mg/ml for phospho-GSK3β and 0.045 mg/ml for total GSK3β ELISAs) in 100 µl kit supplied sample buffer loaded in duplicates to the ELISA microwells. Following a 2 h incubation at 37 °C, wells were washed in kit supplied wash buffer and 100 µl of detection antibody added and samples incubated for 1 h at 37 °C. After washing, 100 µl of reconstituted HRP-linked secondary antibody was added to the wells and incubated for 30 min at 37 °C, followed by incubation with 3,3′,5,5′-Tetramethylbenzidine substrate for 10 min at 37 °C. Upon addition of stop solution absorbance was read at 450 nm using a CLARIOstar multiplate reader (BMG LabTech).

Prior to apoptosis detection 200 000 cells were transfected and seeded into 6-well plates in a volume of 2.5 ml. Three days later total cells were collected and apoptosis was analyzed using the Annexin V-PE/7-Amino-Actinomycin (AAD) apoptosis detection kit (BD Pharmingen). Viable cells with intact membranes exclude 7-ADD and are PE Annexin V negative. Fluorescence generated by the cell-bound Annexin V-PE, which measures the percentage of early apoptotic cells, and the 7AAD, which measures the percentage of late apoptotic cells, were analyzed by FACS. Total apoptosis was calculated by adding the percentage of late apoptotic cells (Annexin V-PE ^High / 7ADD ^High) and the percentage of early apoptotic cells (Annexin V-PE ^High / 7ADD ^Low). Activities of Caspases 3 and 7 were measured using the Luminescent Caspase-Glo 3/7 assay from Promega as described previously [11 (link)], except that luminescence was measured using the CLARIOstar multiplate reader (BMG Labtech).