96 well black polystyrene microplate

Intracellular K⁺ concentrations were determined with a FluxOR II Green Potassium Ion Channel Assay Kit according to the manufacturer's instructions. J774.1 cells were plated in a Corning 96-well black polystyrene microplate. On the day of the experiment, J774.1 cells were first primed with LPS (100 ng/mL) for 5 h before being treated with ATP (5 mM), either in the absence or presence of GSH (5 mM), GSSG (5 mM), or KCl (10 mM) for 30 min. After cells were washed once with DMEM medium, they were loaded with thallium-containing buffer for 1 h in darkness. A high K⁺ stimulus buffer was then added without removing the original buffer. After a 15-min incubation at 20 °C, fluorescent signals were detected by using a Tecan Infinite 200 Pro plate reader (LabX, Midland, ON, Canada), with excitation/emission wavelengths of 485/535 nm, respectively. All reagents in this assay were provided by the FluxOR II Green Potassium Ion Channel Assay Kit.

Cell viability and cytotoxicity were evaluated using the CellTiter-Glo^® Luminescent Cell Viability Assay Kit (Promega, Madison, WI, USA) by following the manufacturer’s instructions. Briefly, MARC-145 cells or PAMs were seeded into 24-well plates at a density of 5 × 10⁴ cells/well or 4 × 10⁵ cells/well and cultured for 24 h. Then, fresh medium containing the PEI at the indicated concentration was added to the cells. After incubation for another 24 h, the cells were lysed by using 1× passive cell lysis buffer (Promega). The cell lysate was transferred into a 96-well black polystyrene microplate (Corning Inc, Corning, NY, USA). CellTiter-Glo^® reagent was added and mixed with the cell lysate. After incubation for 10 min at room temperature (RT), luminescence signal was determined with VICTORX™ X5 Multilabel Reader (Perkin-Elmer Life and Analytical Sciences, Wellesley, MA, USA).

Kinetic membrane fluidity measurements were performed as described previously (48 (link)) with minor modifications. B. subtilis 168CA was grown in medium containing 0.2% glucose. After reaching an OD₆₀₀ of 0.6, cells were stained with 10-µM Laurdan (AnaSpec) for 5 min, washed in Laurdan buffer [phosphate-buffered saline, 0.2% glucose, 1% dimethylformamide (DMF)], and resuspended in the same buffer to an OD₆₀₀ of 0.8. A DMF concentration of 1% was constantly maintained to prevent precipitation of the dye. Sample aliquots of 100 µL were added to 96-well black polystyrene microplates (Corning), and fluorescence was measured at an excitation wavelength of 350 nm and emission wavelengths of 460 nm and 500 nm with 15 nm bandwidth each. After recording the baseline for 5 min, 100 µL of prewarmed Laurdan buffer containing the respective antibiotics was added and measurements were continued for another 30 min. Cells grown in KCl-MHB were washed and resuspended in Laurdan buffer containing 300-mM KCl. General polarization values of Laurdan were calculated according to Wenzel et al. (48 (link)).

Cell viability was determined using PrestoBlue Reagent 10× (Thermo Fisher Scientific; #A13262). MCF7 cells were seeded at 4.7 × 10³ cells/well in 96-well black polystyrene microplates (Corning, NY, USA; #353376) and incubated overnight at 37 °C to allow for cell attachment. The next day, cells were treated with 1, 10, 50, 100, 200 μM of PF-06424439 for 24, 48, 72, and 96 h. At each time point, cells were washed with HBSS (Gibco, Thermo Fisher Scientific; #14025-050) and incubated in the dark at 37 °C with 10 μL of Presto Blue Cell Viability Reagent diluted in 90 μL of fresh complete medium for 90 min. Cell viability was measured at 560 nm (excitation) and 590 nm (emission) using a CLARIOstar^® microplate reader (BMG LABTECH GmbH, Offenburg, Germany). Anti-proliferative activity of PF-06424439 was calculated as 50% inhibitory concentration (IC₅₀) at 72 h and curve-fitting was performed by non-linear regression analysis (log(inhibitor) vs. normalized response) by using GraphPad Prism 9 software.

For quantification of extracellular polymeric substances (EPS), assays were performed in Corning 96-well black polystyrene microplates with clear, flat bottoms, as described previously (63 (link)). After washing steps following overnight incubation, the wells were treated with fixative (3% paraformaldehyde and 0.25% glutaraldehyde in 0.01 M PBS) for 15 min. The wells were subsequently washed and stained with concanavalin A (ConA) conjugated to fluorescein isothiocyanate (FITC; 25 g/ml) for 15 min at room temperature. Each well was subsequently washed in PBS, and the fluorescence at 488 nm was measured with a SpectraMax i3x spectrophotometer plate reader (Molecular Devices). The analysis was repeated in three independent experiments.

DiSC₃(5) stock solutions were prepared at 100 µM in sterile DMSO and stored at −20°C. Measurements were performed according to Winkel et al. (21 (link)) with minor modifications. B. subtilis 168CA was grown in the presence of 50 µg/mL Bovine albumin serum (BSA). After reaching exponential growth phase (OD₆₀₀ 0.3), 1 µM DiSC₃(5) was added to the cells. A dimethyl sulfoxide (DMSO) concentration of 1% was constantly maintained to prevent precipitation of the dye. Antibiotics were added after the fluorescence baseline had stabilized. Measurements were run for 30 min after antibiotic addition. The assay was performed in 96-well black polystyrene microplates (Corning) using a BMG Clariostar Plus plate reader at an excitation wavelength of 610 nm with a bandwidth of 30 nm and an emission wavelength of 675 nm with a bandwidth of 50 nm.

MQ-NCSU cells were seeded in 96-well Black Polystyrene Microplates (Corning, NY, USA) at a viable cell density of 7.5 × 10⁴ cells/well in DMEM cell culture media and incubated at 40 °C and 5% CO₂ in a humidified incubator for 2 h. The cells were then stimulated with two different doses of EVs isolated from TOC: low (5 µg/mL) and high (25 µg/mL) and incubated at 40 °C and 5% CO₂ in a humidified incubator. At 12 h post-stimulation with EVs, phagocytosis was assessed using pHrodo Red Escherichia coli Bioparticles Conjugates for Phagocytosis (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s instructions, with some modifications. Briefly, pHrodo Red Escherichia coli Bioparticles Conjugates were resuspended in 2 mL Live Cell Imaging Solution (Invitrogen, Burlington, ON, Canada) and sonicated to homogeneously disperse the particles. For each well, 25 µL of the medium was removed and replaced with 25 µL of resuspended beads. Untreated macrophage controls were included. The samples were then incubated at 37 °C for 4 h. Finally, fluorescence was evaluated using a fluorescence plate reader at an excitation/emission spectra of 560/586 nm.