Clariostar plus plate reader

All activity assays were performed at 37°C in a buffer containing 32 mM sodium phosphate, 15 mM sodium citrate (pH 5.0), and 100 mM NaCl, and were initiated by the addition of the appropriate amount of enzyme. Kinetic parameters were determined for pNP α-L-fucopyranoside using substrate concentrations between 50 μM and 2 mM. Assays were performed in 96-well-plates and each plate included pNP standards in an identical buffer system for concentration calibration. pNP α-L-Fuc hydrolysis was measured after stopping the assay with a 1:1 ratio of 1 M Na₂CO₃ (pH 11.2). Absorbance at 405 nm was measured with a CLARIOstar Plus plate reader (BMG Labtech). Kinetic parameters for the hydrolysis of fucose-α-1,2-galactose were measured using the L-fucose assay kit (Megazyme; K-FUCOSE) according to the manufacturers recommended procedures for microplate use.

The expression from the PsodB and PsodC promoters was measured using miniTn7T insertions carrying either a PsodB::gfpmut3 or a PsodC::gfpmut3 transcriptional fusion (strains AB5075/miniTn7T-zeo-PsodB::gfpmut3 and AB5075/miniTn7T-zeo-PsodC::gfpmut3, respectively). An AB5075/miniTn7T-zeo-gfpmut3 strain (carrying an empty control), was used as a baseline control. Saturated overnight cultures of the different strains were diluted 1:100 (v/v) in fresh LB broth supplemented with the kaempferol and colistin combination treatment or a DMSO mock treatment. Cultures were incubated for 2 h at 37 °C, 180 rpm. Afterwards, 1 mL samples were washed with PBS and eventually resuspended in PBS. Then, samples were placed in a 96-well plate and their OD₆₀₀ and GFP fluorescence (excitation: 485 nm; emission: 535 nm) were measured in a Clariostar Plus plate reader (BMG LabTech). The fluorescence readings were normalised by their respective OD₆₀₀ and the baseline fluorescence obtained from the empty transposon control was subtracted from those obtained with the strains bearing either the PsodB::gfpmut3 or the PsodC::gfpmut3 the promoter fusions. Three biological replicates (two technical replicates each) were performed for each experimental condition.

HPAC cells were
seeded in U-bottom 96 well-plates pre-treated with 0.5% polyHEMA [poly(2-hydroxyethyl
methacrylate), Sigma in 95% ethanol] at 2 × 10⁴ cells
per well. The plate was then centrifuged at 700 rpm at 37 °C
using a BMG Labtech ClarioStar (plus) plate reader for 10 min. The
cells were incubated for 96 h to allow spheroid formation before any
dye was added for imaging studies. Ru-bqp-ester, Ru-bqp-MPP, and Ru-bqp-R8
were added to the spheroids at 30 and 100 μM in the 96 well
plates and after 24 h incubation, the spheroids were carefully transferred
to an 8-chamber slide (ibidi), with a single spheroid per chamber,
and directly imaged using a Leica TCS DMi8 confocal microscope (40×
oil immersion objective). Hoechst 33342 nuclear stain (1 μg/mL)
was added to the spheroids for 45 min as a contrast agent and excited
using a 405 nm laser with emission collected between 425 and 475 nm.
The Ru(II) complexes were excited using a white light laser at 490
nm and emission collected between 580 and 730 nm. Spheroid images
were acquired using z-scanning across the z-axis
of the samples. On average, 40–50 images were acquired per
z-scan and used to obtain 3D spheroid reconstructions using Leica
Application Suite X (LAS X) software.

Outer cell membrane permeability was measured using a nitrocefin-based leakage assay^{72 (link)} executed as follows. BL21 DE3 cells (encoding chromosomal chloramphenicol resistance, and extrachromosomal penicillin resistance conveyed by beta lactamase on a pet15b plasmid), were grown in a liquid LB medium containing chloramphenicol (30 μM) and carbenicillin (130 μM) to early exponential phase (OD590 = 0.2–0.3). Cells were then centrifuged at 4 °C and 3220 g for 20 min and washed twice with PBS. The cell pellet was re-suspended in 1/10 volume of PBS compared to the original culture volume. 10 μL of the cell suspension was diluted with the 3K peptide solutions to a final volume of 100 μL. Cells were incubated for 20 min in sterile, low-bind, U-bottom 96-well microplates (Greiner Bio-One, Hungary) with oxygen penetrating lid at 37 °C, at continuous shaking in a BioTek Synergy Mx plate reader 436. Following incubation of the cells with the peptide, nitrocefin (1 mg/mL solution freshly diluted with water from a 10 mg/mL DMSO stock solution) was added to a 50 μg/mL final concentration, and the chromogenic hydrolysis of nitrocefin was monitored by reading absorbance at 490 nm every 15 s for 100 cycles in a CLARIOstar plus plate reader (BMG Labtech). Initial velocities of the β-lactamase activity were evaluated from fits to the linear part of the progress curves.

Briefly, we used Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) to transfect the SP1+MCRE-MHI into the 293T cell line. 24 h after transfection, 30000 cells were plated into each well of a black 96 well plate. 48 h after transfection the cells were exposed to FDA Approved Drug Screening Library (L1300-Z298012) via the High Throughput Screening Services, ChemCore Facilities (Johns Hopkins University). Each drug was exposed in 20 μM final concentration. Total well GFP was read with the CLARIOStar Plus Plate Reader (BMG LABTECH, settings: gain 1500, 470–15 excitation and emission 515–20 at focal height 2.6) 16, 28, and 40 h after exposure of the library. Ratio of increased fluorescence was calculated by dividing GFP reads from positive control (SP1+MCRE-MHI transfected but untreated) at each time point with GFP reads of each whole well exposed to drug at the same timepoint for wells in the same lane. For further analysis, we used Excel from Microsoft and GraphPad Prism (v.9).

The number of viable cells was determined using the cell counting kit-8 (CCK-8; Sigma-Aldrich, Merck, kGaA, Darmstadt, Germany, 96992). PC12 cells were plated in a 96-well plate with 1 µM rapamycin (Bio-Techne, Minneapolis, MN, USA, Tocris, 1292, dissolved in DMSO) or vehicle (with 0.0005% v/v DMSO) or 1 µM NBP14 (dissolved in water) or vehicle conditions in full culture media. CCK-8 reagent was added to the wells (10% v/v) 24 or 48 h after plating. The plate was then incubated at 37 °C for 1 h before the absorbance was detected at 450 nm in a CLARIOstar Plus plate reader (BMG Labtech, Aylesbury, UK). For data analysis, each value was represented as a percentage of control to account for inter-assay variability.