Sense beta plus

To evaluate cell death, an assay for the activity of the intracellular enzyme lactate dehydrogenase released into the medium from the dead cells was used [54 (link)]. To this end, a 75 µl aliquot of the culture medium from each well was transferred to a fresh 96-well plate. After that, 10 µl of the following reagents were added to each well: 36 mg/ml of lactate in phosphate-buffered saline, pH 7.2; 2 mg/ml of INT in the diaphorase buffer (see below); 3 mg/ml of NAD⁺ mixed with 6 U/ml diaphorase in 0.03% bovine serum albumin and 1.2% sucrose in phosphate-buffered saline, pH 7.2. The reaction mixture was incubated for 20 min at room temperature, and the optical density of the solutions was determined at the wavelength of 490 nm using the Hidex Sense Beta Plus microplate reader (Hidex, Turku, Finland). The positive control was the cell culture treated with the solvent alone, and the negative control was treated with 0.9% Triton X-100.

This assay was performed according to the Reactive Oxygen Species (ROS) Detection Kit protocol (PromoKine, #PK-CA577-K936). Ma-Mel-19 cells were plated at 20,000 cells per well in their standard cell culture medium in flat bottom 96 well cell culture plates (Corning, 96-well Clear Flat Bottom Polystyrene TC-treated Microplates, #3598) and incubated in a 37 °C 5% CO₂ incubator overnight. The next day, medium was removed, and cells were washed with ROS Assay Buffer, treated with ROS Label (1×), and incubated in a 37 °C 5% CO₂ incubator for 45 min. ROS Label was removed, and cells were washed once more with ROS Assay Buffer. Cells were treated with various amounts of CAP as indicated and were measured 1 h after treatment. The positive control was treated with ROS Inducer (1×) for 1 h prior to measurement. Fluorescence was recorded at Ex 485 nm/Em 535 nm wavelengths on a Sense Beta Plus microplate reader (Hidex, Turku, Finland) with Plate Reader Software Version 0.5.55.0.

CellTiter 96^® aqueous nonradioactive cell proliferation assay (Promega, Madison, WI, USA) was performed to evaluate the in vitro effects of evaluated compounds in breast adenocarcinoma MCF7/S0.5 (parental MCF7 cells adapted to low-sera conditions) and human kidney immortalized cell line HK-2. The employed method uses the bioreduction of tetrazolium salt of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) into a colored formazan with an absorbance peak maximum at wavelength 490 nm. Only viable cells are able to metabolize the compound. Experiments were conducted in accordance with manufacturer guidelines. Briefly, cells were treated with the test compounds, negative control (SDS 10%) or vehicle (DMSO 0.1%) for 48 h in 96-wells plates. At the end of the treatment, 20 μL of MTS reagent was added to each well and incubated for a further 3 h prior to absorbance measurement using a plate reader (Hidex Sense Beta Plus plate reader, Hidex, Turku, Finland). MCF7/S0.5 cell line was cultivated in DMEM/F-12 media w/o phenol red supplemented with 1% FBS and insulin 6 ng/mL. HK-2 cells were cultivated in DMEM with high-glucose and L-glutamine, supplemented with 10% FBS. Results are expressed as the relative cell viability, considering the vehicle to have 100% viability.

Whole cell lysate was prepared with lysis buffer (C2978, Sigma-Aldrich). Three micrograms of PC3 cell whole lysate was used for pyruvate kinase assay (ab83432, abcam) following manufacturer’s instruction. Kinetic measurement at O.D 570 nm by microplate reader (Hidex Sense Beta Plus) recorded pyruvate kinase activity every minute for 40 min.

Reactive oxygen species accumulation was measured using the Fluorometric Intracellular Ros Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s protocol. In brief, after the incubation with the substances, cell medium was removed, and 100 µL of the ROS Detection Master Mix was added to each well and incubated for 30 min in CO₂ incubator at 37 °C. After the incubation, the fluorescence intensity was determined using the microplate reader (Hidex Sense Beta Plus, Hidex, Turku, Finland) at λ_ex = 620 nm, λ_em = 665 nm.

Cells were cultured for two days at 37 °C on black 96-well plates. Immediately before the experiments, cells were incubated with 2 mM Fluo-4AM ester reagent and 1.25 mM probenecid (organic anion transporter inhibitor) for 1 h at room temperature. After the incubation, the cells were washed out with the extracellular solution (140 mM NaCl, 2 mM CaCl₂, 2.8 mM KCl, 4 mM MgCl₂, 20 mM HEPES, 10 mM glucose; pH 7.4). The last washout was supplied with 10 uM α7 nAChR positive allosteric modulator PNU 120596 and tested compound. The excitation of Fluo-4 achieved at 485 nm and fluorescence registered at 535 nm using Hidex Sense Beta Plus (Turku, Finland) multi-well plate fluorimeter. The calcium rise amplitude was measured from the base level of each well.

Protein concentration was determined using the BCA assay [77 (link)]. The following base reagents were used: Reagent A (bicinchoninic acid 1%, Na₂CO₃*H₂O 2%, sodium tartrate 0.16%, NaOH 0.4%, NaHCO₃ 0.95%, pH 11.25), Reagent B (4% CuSO₄*5H₂O), S-WR (50 volumes of Reagent A + 1 volume of Reagent B). 5 µL of cell lysate was mixed with 40 µL of S-WR and incubated for 15 min at 60 °C, after which the optical density was measured at λ = 562 nm using the Hidex Sense Beta Plus microplate reader (Hidex, Turku, Finland). Each sample was assayed in triplicate. Cell lysis buffer was used as a background control. Bovine serum albumin solution in the cell lysis buffer was used as a positive control and to build a calibration curve.