Victor3 multilabel plate counter

Transduced Dubca cells overexpressing human Panx1 were seeded at a cell density of 12,000 cells per well (37,500 cells/cm²) in flat-bottom, 96-well culture plates (Corning, Glendale, AZ, USA) and incubated at 37 °C overnight (5% CO₂). Drugs were dissolved in classic buffer (Tyrode buffer; 124 mM NaCl, 2.44 mM KCl, 10.82 mM NaHCO₃, 0.38 mM NaHP0₄ * H₂O, 0.91 mM MgCl₂ * 6 H₂O, 1.82 mM CaCl₂ * 6 H₂O) and osmotic buffer (Tyrode buffer; 124 mM NaCl, 5 mM KCl, 10.82 mM NaHCO₃, 0.38 mM NaHP0₄ * H₂O, 0.91 mM MgCl₂ * 6 H₂O, 1.82 mM CaCl₂ * 6 H₂O). Following a washout period of 30 min using a classic buffer, cells were preincubated with the drugs in a defined concentration range (Table 1) in a classic buffer for 15 min. Cells were subsequently exposed to the same range of concentrations of each drug (Table 1) in an osmotic buffer for 30 min to trigger the opening of Panx1 channels via osmotic shock. Throughout the procedure, cells and solutions were kept at 37 °C in an incubator (5% CO₂). Next, 50 µL of each solution was transferred into white, opaque, 96-well plates (Corning, Glendale, AZ, USA), and extracellular ATP release was measured using an ATP Bioluminescent Assay Kit, following the manufacturer’s instructions (Sigma-Aldrich, Overijse, Belgium), in a VICTOR3 Multilabel Plate Counter (PerkinElmer, Waltham, MA, USA).

Cell viability was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich, Overijse, Belgium) assay. Transduced Dubca cells overexpressing human Panx1 were seeded in flat-bottom, 96-well culture plates (Corning, Glendale, AZ, USA) at a cell density of 12,000 cells/well (37,500 cells/cm²). Then, 24 h after seeding, cells were exposed to the drugs dissolved in 200 µL of phenol red-free DMEM (Gibco, Waltham, MA, USA), supplemented with Glutamax^TM (Gibco, Waltham, MA, USA), 10% FBS (Gibco, Waltham, MA, USA), streptomycin, and penicillin (Thermo Fisher Scientific, Waltham, MA, USA) for 24 h or in classic buffer (Tyrode buffer; 124 mM NaCl, 2.44 mM KCl, 10.82 mM NaHCO₃, 0.38 mM NaHP0₄ * H₂O, 0.91 mM MgCl₂ * 6 H₂O, 1.82 mM CaCl₂ * 6 H₂O) for 45 min to a predetermined concentration range of each drug (Table 1). Cells were washed with 100 µL of warm phosphate-buffered saline (PBS) and incubated with MTT solution (0.5 mg/mL MTT in phenol red-free DMEM media) for 1.5 h. Next, each well was washed with 100 µL of PBS, and the water-insoluble formazan crystals were dissolved using 100 µL DMSO. After shaking for 10 min, absorbance was measured using a VICTOR3 Multilabel Plate Counter (PerkinElmer, Waltham, MA, USA) at a wavelength of 570 nm. Viability was expressed relative to the untreated control cells.

Cell viability was assessed by means of an MTT assay for two purposes. First, inhibitory concentrations of the different chemicals that reduce cell viability by 10%, namely IC₁₀ concentrations, were established with an MTT assay during earlier performed in vitro carcinogenicity testing in human hepatoma HepaRG cell cultures [30 (link)]. Second, an MTT assay was used in this study to determine cell viability after measurement of connexin hemichannel-related ATP release (see 2.8). For the latter purpose, cells were washed with PBS after exposure to the chemicals and incubated for 90 min at 37 °C with an MTT solution (0.5 mg/mL MTT in Williams’ E medium (Thermo Fisher Scientific, Waltham, MA, USA)). After incubation, the MTT solution was removed and 100 µL DMSO was added to each well to dissolve the formed formazan crystals. Plates were shaken for 10 min and absorption was measured at 595 nm on a VICTOR³ Multilabel Plate Counter (PerkinElmer, Waltham, MA, USA). Data were expressed as a ratio to the solvent control cells (DMSO CTL or PBS CTL depending on the respective solvent of the compound; cells exposed to medium containing 0.5% v/v DMSO or PBS). The use of DMSO or PBS as a solvent was based on preceding in vitro testing [30 (link)].

ELISA was applied for additional analysis of RANKL expression. Serum RANKL levels were measured using (ab100749 RANKL Mouse Elisa kit, Abcam). The optical density was measured using Victor³ Multilabel plate counter (Perkin Elmer) at 450 nm, and the result was calculated using standard curve on a log–log scale.

Cry1Ab insecticidal protein was incorporated into artificial diets by adding freeze-dried material from the leaves of commercial Bt maize DKC6667Y (Cry1Ab) MON810 while non-Bt maize DKC6666 (isogenic) was added to the non-Bt diets. Leaves from Bt and non-Bt maize had been collected in the summer of 2017 in field areas near Lleida in unsprayed plants. Leaves were cut into small strips and the main veins were removed. The material was later freeze-dried in a vacuum drier (Gamma 2–16 LSC plus, CHRIST, Osterode am Harz, Germany) and ground in a Thermomix^® until a fine powder was obtained. The lyophilized material was kept at −80 °C until use. The content of Cry1A insecticidal protein in Bt and Bt-β diets was verified using the Agdia Bt-Cry1Ab/Cry1Ac kit (Agdia Inc., Elkhart, IN, USA). For calibration, Cry1Ab standards at 100, 75, 50, 25, 15, 8, and 2 ng/mL were used. Measurements were made with a VICTOR3 Multilabel Plate Counter (PerkinElmer Life and Analytical Science, Madrid, Spain) at 650 nm.
ELISA analyses indicated that the toxin content in Bt diets amounted to 9 µg of Cry1Ab protein per gram of diet. This amount is similar to the average toxin content in a maize plant developed in the field [26 ].