Victor2 multilabel plate reader

After 72 h of transfection, AtT-20 cells were trypsinized, counted and re-seeded in a 24-well plate at a density of 12.5 × 10⁴ cells/well in 500 µL of culture medium at 37 °C. The following day cells were incubated with or without pasireotide 10 nM for 4 h [27 (link)]. Murine ACTH levels were determined in cell culture media using a specific Elisa immunoassay kit (Fine Test, Wuhan Fine Biotech Co., Ltd., Wuhan, China), according to the manufacturer’s instructions. Absorbance was read at 450 nm in a Victor2 multilabel plate reader (Perkin Elmer, Waltham, MA, USA). Data were plotted and analyzed with Curve Expert 1.4 program. Hormone levels were normalized on the protein content, measured by BCA assay. The assay was done in triplicate for each condition and experiments were replicated 3 times. Primary cells were transfected for 96 h and stimulated with or without pasireotide 10 nM for 4 h. Human ACTH in culture media was measured by specific chemiluminescent immunometric assay (Immulite 2000, Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA) with an inter-assay coefficient of variation ranging from 6.1 to 10.0%, an intra-assay coefficient of variation ranging from 6.7 and 9.5% and sensitivity of 5 pg/mL.

Protein Assay dye reagent (Bio-Rad) was diluted with 4 volumes of distilled, deionized water. One volume of biological sample (diluted 10- and 20-fold) or bovine serum albumin protein standard was mixed with 50 volumes of diluted dye reagent and 200 µL was loaded into 96-well plate (Thermo Fisher Scientific, #269620). Optical density at 600 nm was recorded with a Victor2 Multilabel plate reader (PerkinElmer). Total protein concentration was interpolated from bovine serum albumin standard curve made with 11 dilution points from 100 mg/mL and 2-fold dilution path. Protein concentration was expressed as mg/mL.

To evaluate (R)-1, (S)-1, (R)-5 and (S)-5 activity, the cells were treated for 24 h without (control) or with concentrations between 1 nM–5 mM (1 nM, 5 nM, 10 nM, 50 nM, 100 nM, 500 nM, 1 µM, 5 µM, 10 µM, 50 µM, 100 µM, 500 µM, 1 mM and 5mM) of the test samples. The culture medium was removed and the cells were further incubated for 2 h with 0.2 mg/mL MTT in PBS. After removal of the medium, the cells were lysed with 0.1 mL of iso-propanol. The absorbance of the solubilized formazan pellet at 540 nm was determined by Victor2, Multilabel plate reader (Perkin Elmer, MI, Italy). The IC₅₀ was determined from three different experiments of the dose-response curve by using GraphPadPrism 5 (GraphPad Software, Inc., La Jolla, CA, USA) fitting a symmetrical sigmoidal shaped curve.

Mouse 3T3‐L1 preadipocytes were purchased from DS Pharma Biomedical. Preadipocytes were cultured in Dulbecco's modified Eagle's medium–high glucose medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% calf bovine serum (Gibco) and penicillin–streptomycin (Nacalai Tesque) at 37°C in a humidified 5% CO₂ atmosphere until confluence was reached in a 96‐well plate format. Two days after confluence (day 0), cells were stimulated to differentiate by culturing in adipocyte differentiation medium (ADM; DS Pharma Biomedical) for 3 days. Cells were then maintained in adipocyte maintenance medium (DS Pharma Biomedical) for an additional 4 days. Extracts or fractions (10 μg/ml) were administered from day 0 of adipocyte differentiation. On day 7, intracellular lipid droplets were stained using AdipoRed Assay Reagent (Lonza) according to the manufacturer's instructions. After obtaining images using a BZ‐X710 fluorescence microscope (Keyence), intracellular lipid accumulation was quantified by measuring fluorescence (Ex 485 nm/Em 590 nm) using a Victor2 multilabel plate reader (PerkinElmer).

To detect prolactin (PRL) and adrenocorticotrophic (ACTH) hormone levels specific Elisa immunoassay kits (Fine Test, Wuhan Fine Biotech Co., Ltd, Wuhan, CN) were used.
MMQ and AtT-20 cells were seeded in six-well plate, at a density of 3,3 × 10⁵ cells/well. After 24 h cells were transfected with wild-type, S2152A and S2152D FLNA plasmids for 72 h at 37°C. Then cells were counted and re-seeded in a 24-well plate at a density of 12,5 × 10⁴ cells/well, in 300 μl of complete medium for AtT-20, while MMQ were re-seeded in a six-well plate at a density of 3,6 × 10⁵ cells/well, in 1 ml of complete medium. Cells were than incubated with or without BIM53097 100 nM for 4 h (AtT-20) or 24 h (MMQ), based on preliminary time course experiments. After treatment, culture media were collected to perform the assay, according to the manufacturer’s instructions. Absorbance was read at 450 nm in a Victor2 multilabel plate reader (Perkin Elmer, Whaltam, MA, USA). Data were plotted and analysed with the specific Curve Expert 1.4 program. Hormone detection were done in triplicate and experiments were replicated three times for each cell line. Hormone levels were normalized on the protein content, measured by BCA assay. Western blot analysis was carried out before each experiment to test transfection efficiency

The cytotoxicity of UAMC-3203 was assessed by MTT assay. The HCE cells were seeded on a 96-well plate at a density of 100,000 cells/well in 200 µL of supplemented growth medium. The next day the cells were washed with PBS (pH 7.2). The cells were then exposed to UAMC-3203 at concentrations 10 nM, 1 µM, 10 µM, and 50 µM for 3 h. For control, cells not exposed to UAMC-3203 cultured in a serum-free medium were used. The cells were then washed twice with PBS and incubated 2 h with 100 µL of 10% of thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO, USA) in a serum-free medium. An amount of 100 µL of 20% (w/v) sodium dodecyl sulfate (SDS), N-dimethylformamide (DMF) lysis buffer was added to each well and incubated overnight. The following day, cell viability was evaluated by measuring absorbance at 570 nm with a Victor² multilabel plate reader (PerkinElmer, Wallac, St. Paul, MN, USA). The cell viability % was calculated as below: $$\% of cell viability=\frac{\left(Abs exposed cells-Abs blank\right)}{\left(Abs non-exposed cells-Abs blank\right)}\times 100$$
where Abs exposed cells = absorbance of cells exposed to UAMC-3203; Abs blank = absorbance of well plates without cells; and Abs non–exposed cells = Absorbance of cells exposed to serum-free medium (i.e., control).

Organoids were mechanically dissociated by vigorous pipetting. 20–100 fragments were seeded in 10 µl BME in a 24-well plate. 24 h after seeding, media was removed and replaced with fresh media containing either 5 μM Kras4B vivo-morpholino (Genetools - GTATAGAAGGCATCGTCAACACCCT) or 5 μM control vivo-morpholino (Genetools - CCTCTTACCTCAGTTACAATTTATA). Cell proliferation was assessed after 6 days later by adding 10% Resazurin (R&D systems, AR002). Fluorescence was measured using Victor2 Multilabel Plate Reader (PerkinElmer) as already described.