Victor3 multilabel reader

Shortly before the sacrifice, blood was collected by intracardiac puncture with stratification across groups to reduce the potential impact of circadian rhythm and pulsatility. Blood samples were left to coagulate at room temperature for 1 h, centrifuged for 15 min at 2000 rpm twice in a cooled bench-top centrifuge (Microlite Microfuge, Thermo Electron Corporation) and stored at -80 °C until use. Serum levels of all hormones were measured in the same analytical section by the following commercial ELISA kits of the same lot(s):

E2 Rat kit (RTC009R—BioVendor Brno, Czech Republic), LOD 2.5 pg/ml

T Mouse/Rat kit (RTC001R—BioVendor Brno, Czech Republic), LOD 2.5 pg/ml

TSH Rat Kit (ELK2283—ELK Biotechnology, China), LOD 0.071 ng/mL

T4 Rat Kit (ELK8716—ELK Biotechnology, China), LOD 1.42 ng/mL

Each kit provided a standard solution of the hormone and serial dilutions were prepared to derive a standard curve and define the range of linearity of each test. For all the analyses, the manufacturer’s instructions were followed. Each sample was assessed in duplicate and the absorbance was read at 450 nm on a VICTOR3 Multilabel reader (Perkin Elmer, USA). The unknown hormone concentrations in samples were derived using the standard curve of each hormone and the software GraphPad Prism 5.0 (GraphPad Software Inc.).

MCF-7 cell viability was assayed by the MTT assay as described previously [53 (link)]. Cells were seeded in 96-well plates and grown to confluence. Then, the medium was removed, fresh medium was added, and the cells were returned to the incubator in the presence of different concentrations of clotrimazole (0–100 μM). After 24 h, cells were incubated with 5 mg/mL MTT reagent (3,4,5-dimethiazol-2,5-diphenyltetrazolium bromide, Sigma—Aldrich Co., St. Louis, MO, USA) for 3 h. Thereafter, the formazan crystals were dissolved in DMSO, and the absorbance at 560 nm was evaluated in a VICTOR3 multilabel reader (PerkinElmer, Waltham, MA, USA) with the subtraction of background absorbance at 670 nm [53 (link)].

EGFR kinase inhibition was determined using the Z´-LYTE^™ kinase assay kit-Tyr 4 peptide (Invitrogen, PV3193). Briefly, EGFR kinase (Invitrogen, PV3872) was dissolved in a reaction buffer composed of 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl₂ and 1 mM EGTA. The final 10 μL of the kinase reaction mixture consisted of 5 ng kinase, 2 μM substrate peptide Try4, ATP and the test compounds theliatinib, gefitinib and erlotinib. ATP (10, 50, 200, 500, 800 and 1000 μM) was finally added to the reaction mixture to initiate the enzymatic reaction. The final concentration of DMSO in the assay was 2%. The reaction mixture was incubated at 25°C for 60 minutes in a 384 well plate. Then, 5 μL of Development Reagent B was added per well and incubated for further 60 minutes at 25°C. The fluorescent signal was read at emission wavelengths, 445 nm and 520 nm after excitation at 400 nm in a Victor3 multi label reader (PerkinElmer). The kinetic parameters (Ki, V_max and Km) were calculated with the Graphpad Prism software according to the Michaelis-Menten equation: V = Vmax × [S]/ (Km×(1+[I]/Ki)+ [S]). IC₅₀ of thliatinib, gefitinib and erlotinib was calculated at different ATP concentrations using XLfit software (IDBS, Guildford, UK) [29 ].

The ROS Detection Assay Kit (BioVision, Milpitas, CA, USA) was used to measure the amount of intracellular ROS following the manufacturer’s protocol. Briefly, 10,000 HepG2 or A549 cells in 100 µL of culture medium/well were seeded on 96 flat-bottomed multi-wells and incubated overnight at 37 °C to allow adhesion. After 24 h, the medium was removed and cells were washed once with 100 μL ROS assay buffer, combined with 100 μL/well 1X ROS assay label, and incubated for 1 h at 37 °C. At the end, the ROS label solution was removed and cells were treated with the three highest concentrations of MZ (at 295.7, 29.6 and 2.96 μM), ZOX (at 463.4, 46.34 and 4.63 μM), vehicle as negative control (DMSO 0.8% and 0.2% for cells treated with MZ and ZOX, respectively), or H₂O₂ 100 μM as positive control in duplicated wells, and incubated for 24 h at 37 °C. Plates were read for green fluorescence from the bottom (485 nm excitation, 535 nm emission) by the Victor 3 Multilabel Reader (PerkinElmer). Three independent experiments were performed. After background subtraction, the fold-change of the fluorescence reading of treated cells with respect to vehicle control cells was calculated.

Cells were seeded into 96-well plates at a density of ~1 × 10⁴ cells/well and incubated at 37°C with 5% CO₂ for 24 h. Following incubation, media were removed from the plate and the cell monolayer was treated with the selected probiotic strain at ~10⁵ or ~10⁷ CFU/well in DMEM without antibiotics for 12 h. As for the case cell-free supernatant (CFS) of the probiotic bacteria, the RAW 264.7 cell monolayer was treated with 10% or 20% of CFS in DMEM for 12 h. Following incubation, RAW 264.7 cells plus probiotics were exposed to 2,000 ng/ml lipopolysaccharide (LPS) for 24 h. After incubation, the production of nitric oxide (NO) was measured using Griess reagent (Sigma Aldrich, USA) following the manufacturer’s instructions. Briefly, 50 μl of the culture supernatant was transferred to a new plate. Then, 50 μl of Griess reagent was added. After 1 min of mixing in a shaker, the plate was incubated at 37°C for 15 min. Absorbance was measured at 540 nm using a Victor ×3 Multilabel Reader (Perkin Elmer 2030).