Fluoronunc

Quantification of the H-ficolin concentration was performed blinded to subject identity as previously described using normal human serum as standard except for a few changes^{39 (link)}. In brief, serum was thawed, diluted in assay buffer, and added to microtiter wells (FluoroNunc, Thermo Scientific, Waltham, MA, USA) coated with acetylated bovine serum albumin (B2518, Sigma-Aldrich, St. Louis, MO, USA), which is recognized by H-ficolin. Standards, samples, and controls were added automatically to plates using a Janus Varispan automated work-station (PerkinElmer, Waltham, MA, USA). In-house biotinylated anti-H-ficolin antibody, europium-labelled streptavidin (PerkinElmer) and enhancement solution (Ampliqon, Odense, Denmark) were added in successive steps with triple washing in between. The europium fluorescence intensity was detected with a Victor X5 fluorometer (PerkinElmer). Intra-assay and inter-assay coefficients of variation were below 10% and 16%, respectively. H-ficolin concentration was used as a predictor as a continuous variable as well as by comparing subjects grouped according to H-ficolin quartiles.

The effects of nicotine on tubulin polymerization were assessed using the fluorescence‐based tubulin polymerization assay kit according to the manufacturer's instructions (Cytoskeleton, Denver, CO). Briefly, a buffer containing 2 mg/mL porcine brain tubulin, 80 mM PIPES pH 6.9, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP, and 15% v/v glycerol was incubated with either 0.01–10 mM nicotine, 2.3 μM colchicine, or 3 μM paclitaxel in black, clear/flat‐bottom 96‐well plates (FluoroNunc™, Thermo Fisher Scientific) within a fluorimeter (FlexStation® 3; Molecular Devices, San Jose, CA) pre‐warmed to 37°C. Excitation and emission wavelengths of 360 and 420 nm, respectively, were used, and readings were taken every minute for a total of 60 min. Maximum velocity (V_max) and maximum product values were subsequently extracted from the data using SoftMax® Pro software (v7.0 GxP, Molecular Devices).

Drug susceptibility of Mab was determined using the REMA method [33 (link)]. Log-phase cultures were diluted at concentrations of approximately 10⁶ bacteria/mL. Then, 100 μL of the bacterial suspensions was added in a 96-well black plate (Fluoronunc, Thermo Fisher, Waltham, MA, USA) containing 100 μL of Middlebrook 7H9, without the addition of Tween 80, in the presence of serial compound dilution. Growth controls containing no compound and sterile controls without inoculum were also included. A volume of 10 μL of resazurin (0.025% w/v) was added to each well after 24 h, and bacterial viability was assessed after a further 18–24 h of incubation using a FluoroskanTM Microplate Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA; excitation = 544 nm, emission = 590 nm). Bacterial viability was calculated as a percentage of resazurin turnover in the absence of compound.
MICs of the clinical isolates were determined by means of the micro-broth dilution method. Dilutions of clinical isolates (about 10⁶ CFU/mL) were streaked onto 7H11 solid medium containing a range of drug concentrations. Plates were incubated at 37 °C for about 5 days for MABSC or 7 days for MAC. The growth was visually evaluated: the lowest drug dilution at which visible growth failed to occur was taken as the MIC value. Results were expressed as the average of at least three independent replicates.

SYTOX assays were conducted with suspensions of PAO1 resting cells (≈10⁷ CFU/mL) prepared from an actively growing culture (OD₆₀₀ ≈ 0.4–0.6) pelleted (3000× g, 10 min, 20 °C) and resuspended in 20 mM NaPiB, pH 6.0, 150 mM NaCl, 100 mM sorbitol. 100 µL of this suspension were added to each well of a FluoroNunc 96-well plate together with 5 µM of the probe, and fluorescence was recorded in a Varioskan Flash microplate reader (Thermo Fisher Scientific, Waltham, MA, USA) with an excitation wavelength of 485 nm and 520 nm for the fluorescence detection. Incubation was prolonged until baseline was reached and then 100 µL of the compounds assayed at the proper concentration were added to each well. Then, measurements were prolonged for 60 min at 37 °C. NPN assay was similar, but the bacterial suspension was at 10⁸ CFU/mL culture, and the final probe concentration was 10 µM. The fluorescence recording settings were 350 nm excitation, and 420 nm emission.