M200 pro infinity plate reader

A Toxilight assay from Lonza (Basel, Switzerland) was used to assess membrane toxicity according to the manufacturer’s protocol. The release of adenylate kinase (AK) was measured in the medium, reflecting the integrity of the plasma membrane. H9c2 cells were treated for 24 h with 1 to 100 μM imatinib and sorafenib in the presence of glucose or galactose media. After this incubation time, we removed 20 μL of supernatant from each well and then transferred the supernatant into a new opaque 96-well plate. An amount of 100 μL of assay buffer was then added to each well. After 5 min incubation, luminescence was measured using a Tecan M200 Pro Infinity plate reader (Männedorf, Switzerland). A volume of 0.1% Triton X was used as a positive control. All data were normalized to positive control incubations containing 0.1% Triton X (set at 100% cell lysis).

Mitochondrial superoxide was assessed using MitoSOX Red (Invitrogen, Basel, Switzerland) according to the manufacturer’s manual. H9c2 cells were seeded into black costar 96-well plates (20,000 cells/well). H9c2 cells were treated with 1 to 100 μM imatinib and sorafenib for 24 h in the presence of glucose or galactose media. The incubation with 100 µM antimycin A for 30 min was used as a positive control. After 24 h of exposure, cell culture medium was removed, and 2.5 µM MitoSOX dissolved in 100 µL Dulbecco’s phosphate-buffered saline (D-PBS) was added. After incubation for 10 min at 37 °C in the dark, fluorescence was measured (excitation, 510 nm; emission, 580 nm) using a Tecan M200 Pro Infinity plate reader (Männedorf, Switzerland). We normalized the results to the protein content using a Pierce Bicinchoninic acid protein assay kit (Thermo Fischer Scientific, Darmstadt, Germany) according to the manufacturer’s instructions. Results were normalized to the 0.1% DMSO control condition.

Myoblasts (5,000 cells/well) were seeded in a 96-well plate and differentiated to myotubes. Myotubes were then exposed to 10 μM simvastatin and/or 10 ng/mL or 100 ng/mL insulin for 24 hours. We used Triton-X 1% as a positive control, which destroys cells and depletes the intracellular ATP content completely. Intracellular ATP was determined using the CellTiterGlo Luminescent cell viability assay (Promega, Switzerland), in accordance with the manufacturer’s instructions. Briefly, 100 μL of assay buffer was added to each 96-well containing 100 μL culture medium. After incubation for 10 minutes, luminescence was measured using a Tecan M200 Pro Infinity plate reader (Männerdorf, Switzerland).

Changes in the intracellular ATP content were measured using the CellTiter-Glo^® kit from Promega (Dübendorf, Switzerland) according to the manufacturer’s protocol. SH-SY5Y cells were treated as described above. After 6 and 24 h of treatment, 80 μL of assay buffer was added to each well containing SH-SY5Y cells in 80 μL of culture medium. The plate was shaken for 2 min at 350 rpm, followed by 15 min of incubation at RT. The ATP content was determined by luminescence measurement using a M200 Pro Infinity plate reader (Tecan, Männedorf, Switzerland). All data were normalized to DMSO 0.1%-treated cells (control).

As marker of membrane toxicity, cell membrane integrity loss expressed as adenylate kinase (AK) release, was determined with the ToxiLight BioAssay kit (Lonza, Basel, Switzerland) [38 (link)]. In brief, C2C12 myoblasts were seeded at a density of 50,000 cells per well in a 96-well plate. After attachment, the cells were treated with 100 μL of culture medium containing the test drugs (naphyrone at the concentrations of 10–500 μM, the remaining drugs at concentrations of 50–2000 μM). Triton X-100 (0.1%) was included as positive control inducing cell lysis. After 24 h, 20 μL of cell supernatant was transferred to a luminescence compatible 96-well plate and 100 μL of AK detection reagent was added to each well. After shaking of the plate at room temperature for 5 min, luminescence was measured with a Tecan M200 Pro Infinity plate reader (Tecan, Männedorf, Switzerland). The percentage of intact cells (no cell membrane integrity loss) was calculated in relation to untreated cells and cells treated with Triton X-100, which were determined to represent 100% and 0% intact cells, respectively.

The intracellular ATP content representing the metabolic cell activity was measured using a CellTiter-Glo kit (Promega, Dübendorf, Switzerland) [38 (link)]. C2C12 myoblasts were seeded at a density of 50,000 cells per well in a 96-well plate. After attachment, the cells were treated with 100 μL of culture medium containing the test drugs (naphyrone at the concentrations of 10–500 μM, the remaining drugs at concentrations of 50–2000 μM). Triton X-100 (0.1%) was included as positive control inducing cell lysis. After 24 h, 50 μL of supernatant was removed from each well before 50 μL of CellTiter-Glo Reagent was added to the remaining cell culture medium at a ratio of 1:1 (v/v). After shaking of the plate at room temperature for 15 min, luminescence was measured with a Tecan M200 Pro Infinity plate reader (Tecan, Männedorf, Switzerland).

Generation of mitochondrial ROS was assessed using MitoSOX Red (Invitrogen, Basel, Switzerland). HepG2 cells were seeded into black costar 96-well plates and exposed to a range of TKIs. The positive control was 100 μM amiodarone. After 48 h, cell culture medium was removed and 2.5 μM MitoSOX dissolved in 100 μl DPBS was added. After incubation for 10 min at 37°C in the dark, fluorescence was measured (excitation 510 nm, emission 580 nm) using a Tecan M200 Pro Infinity plate reader (Männedorf, Switzerland).