96 well microplate

The isolation of satellite cells from the kursiv M. rhomboideus of 10 female five- and 20 days old piglets and the establishment and validation of two muscle cell pools (P5, n = 10; P20, n = 10) were performed as previously described (Metzger et al., 2020 ). For proliferation experiments cells from both pools stored in liquid nitrogen were defrosted and cultured for 72 h at 35°, 37° (control), 39° or 41°C in growth medium with one medium change after 48 h as described by Metzger et al. (2021) (link). A total of 1 × 10⁶ cells from each pool were seeded in 100-mm gelatin-coated culture dishes (Sarsted, Nümbrecht, Germany) for microarray analysis. To explore mitochondrial and glycolytic functional changes, 2,000 cells/well and 20 wells per pool per replicate (Seahorse XFp plate, OLS, Bremen, Germany) were used. To estimate the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG), 3,000 cells/well and 10 wells per pool per replicate were used (96 well-microplates, Sarstedt). Three replicates were generated for each experiment.

The MICs for vancomycin (vancomycin hydrochloride from Streptomyces orientalis with a potency ≥ 900 μg/mg from Sigma-Aldrich, St. Louis, MO, USA) and teicoplanin (from Actinoplanes teichomyceticus with a purity ≥ 80% purchased from Sigma-Aldrich) were determined according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2014 ). In summary, the antibiotics were serially diluted in twofold steps (from 100 to 0.037 mg/L) in 96-well microplates (Sarstedt, Inc., Newton, MA, USA) in MHB. To 150 μL of medium containing the antibiotic in each well, 50 μL of an exponentially growing cell culture diluted to 0.5 McFarland standard was added. The microplates containing S. aureus cells were incubated at 37°C. The MIC was determined for each antibiotic, after 16 h of exposure, by visual inspection and by measuring the optical density of cell cultures in a SpectraMax^® Plus 384 Microplate Reader spectrophotometer (Molecular Devices, Silicon Valley, CA, USA) at 600 nm. At least two independent assays were performed. The MIC was defined as the lowest antibiotic concentration able to inhibit visible bacteria growth after 16 h of incubation at 37°C.

To determine the half-maximal inhibitory concentration (IC₅₀) and to verify the antiproliferative effect of ZK-CH-11d, the MTT colorimetric assay was used. Cells (MDA-MB-231, MCF-7, and MCF-10A) were seeded in 96-well microplates (SARSTEDT, Nümbrecht, Germany) at a density of 5 × 10³ cells per well. Twenty-four hours after cells seeding, the chalcone was added at concentrations of 100, 50, and 10 µmol/L and chloroquine at concentrations of 5, 10, 20, 50, and 100 µmol/L. After 24, 48, and 72 h of chalcone and chloroquine treatment, the cells in each well were incubated for 4 h with 10 μL of MTT (5 mg/mL, Sigma-Aldrich Chemie, Steinheim, Germany) at 37 °C in the dark. During this incubation, MTT was metabolized to insoluble formazan by mitochondrial oxidoreductases in cells. 100 μL of SDS (10% sodium decyl sulfate) was added to each well to dissolve the formazan crystals, and the cells were incubated for another 12 h. An automated Cytation™ 3 Cell Imaging Multi-Mode Reader (Biotek, Winooski, VT, USA) was used to measure absorbance at 540 nm. Three independent experiments were performed for each test.

Plasmin and plasminogen-derived activity in the milk samples were measured as described by [13 (link)]. After thawing the milk, each sample was analyzed in duplicate on 96-well microplates (Sarstedt, Nümbrecht, Germany). In brief, plasmin and plasminogen were dissolved from casein micelles by the incubation of defatted milk with ε-amino-n-caproic acid, followed by ultracentrifugation for 1 h (Optima MAX-XP, Beckman Coulter, Inc., Bromma, Sweden) at 4 °C and 100,000× g. Plasmin activity was measured in the resulting milk serum using 2.5 mg/mL of a chromogenic substrate pyro-Glu-Phe-Lys-p-nitroanilide hydroxy chloride (Biophen CS-41(03), Hyphen BioMed, Neuville Sur Oise, France). Plasminogen, i.e., the inactive precursor of the enzyme, was converted into plasmin after activation with urokinase (49.5 Plough units) to measure total activity, i.e., the sum of plasmin and plasminogen-derived activity. Both total and plasmin activity were measured using a multimode microplate reader (POLARstar Omega, BMG Labtech, Ortenberg, Germany) at 37 °C. Absorbance was recorded every 3 min for 120 min, and activity was expressed as change in absorbance at 405 nm per unit time (ΔA405/Δt). Omega Data analysis software (version 5.50 R4) was used for evaluation of the data. Plasminogen-derived activity was finally calculated as the difference between total activity and plasmin activity.

Cultures in nutritive medium were set in 96-well microplates (Sarstedt, Nümbrecht, Germany), with a final volume of 200 µL per well. The test bacteria were adjusted to 5 × 10⁵ CFU/mL, and 1/2 serial dilutions of the stock suspensions of the AgNPs were distributed in the wells. The corresponding negative controls were also set. Microplates were incubated overnight at 37 °C, in the same microtiter plate incubator/reader indicated above. Absorbance at 660 nm was registered every hour, after 20 s shaking. All samples were implemented at least in triplicate. MIC was determined as the AgNPs concentration in the well with no apparent growth. For MBC determination, 50-μL aliquots of cultures in the wells corresponding to the MIC and two concentrations immediately above were plated in nutritive agar in duplicate. After overnight incubation at 37 °C, the MBC was calculated after colony counting and determination of the concentration of AgNPs corresponding to a killing of at least 99.9% of the original viable bacteria. IC₅₀ values were calculated using GraphPad Prism VIII (GraphPad software, San Diego, CA, USA) from the growth inhibition data corresponding to the incubation times in which the growth controls reached the early stationary phase.

First, 0.05% Trypsin-EDTA (1X) (Gibco, Sigma-Aldrich Company Ltd., Poole, UK), phosphate-buffered saline (PBS) solution tablets (prepared by dissolving one tablet in 200 mL of deionised water), Dulbecco’s Modified Eagle Medium (Gibco DMEM 1X, Sigma-Aldrich Company Ltd., Poole, UK), containing 4.5 g/L of glucose and 0.11 g/L of sodium pyruvate stored at 4 °C, antibiotics prepared with 100 µg/mL of streptomycin sulphate, 0.25 µg/mL of amphotericin B, and 100 U/mL penicillin G sodium, Gibco foetal bovine serum (FBS), thiazolyl blue tetrazolium bromide (MTT) powder, DMSO, menadione, DPPH (1,1-diphenyl-2-picrylhydrazyl), methanol, fluorescein diacetate (FD), propidium iodide (PI), and ascorbic acid were all purchased from Sigma-Aldrich (Poole, UK). L-glutamine, Gibco trypan blue stain (0.4%), 96-well microplates, 24-well microplates, and fluorescence-activated cell sorting (FACS) tubes were purchased from Sarstedt (Sarstedt Ltd., Nümbrecht, Germany). A ROS-Glo™ H2O2 Assay Kit was purchased from Promega (Promega UK Ltd., Southampton, UK). The antioxidant scavenging activity was measured by the use of a microtiter plate reader (Infinite 200 Pro, Tecan Trading, Männedorf, Switzerland). All other materials were obtained from Thermo Fisher Scientific (Loughborough, UK) and used as received.