Versamax absorbance microplate reader

The MIC assay was performed following the guidelines from the Clinical and Laboratory Standards Institute with slight modifications (16 (link)). Briefly, P. aeruginosa was incubated at 37°C overnight and diluted in cation-adjusted Muller-Hinton broth (CAMHB) to approximately 1 × 10⁶ CFU/mL. Bacteria were incubated with 2-fold diluted carbapenems or peptides in 96-well plates (Greiner Bio-One, Frickenhausen, Germany) at 37°C overnight. After incubation, the final OD₆₀₀ was measured using the VersaMax absorbance microplate reader (Molecular Devices, CA, USA), and the minimal inhibition concentration was determined by the concentration of wells at which no bacterial growth was detected. The assay was performed in triplicate, with each sample performed in triplicate wells.

Total protein extract was isolated using 1X Radio-Immunoprecipitation Assay buffer (RIPA; Sigma) supplemented with ethylene-di-amine-tetra-acetic-acid (1%; EDTA, Sigma) and protease inhibitor cocktail (1%; Sigma). Total protein concentration was quantified using Protein Assay Reagent (Bio-Rad, Hercules, CA) and VersaMax absorbance microplate reader (595nm, Molecular Devices). The 80μg of total protein was loaded in each well. Changes in protein expression were quantified by immunoblotting for CFTR (596-ab; CF Foundation), NF-κB (Santa Cruz Biotechnology, Santa Cruz, CA), or β-actin (Sigma) antibodies. The secondary antibodies were anti-mouse (Amersham, Amersham, UK) and anti-rabbit (Amersham).

4T1-luc2 cells were plated in a 96-well plate (Costar) in 100 μl medium at a density of 10,000 cells per well and left over night to adhere. The next day, the medium was replaced with medium containing the experimental compounds: HQ4, HQ4-DTPA, HQ5, and Gambogic acid (GA), three wells per condition. After 24 h, cell viability was measured using a nonradioactive colorimetric MTS viability assay (Promega Benelux) according to the manufacturer’s protocol. Optical absorption was measured at 490 nm with a Versamax absorbance microplate reader (Molecular Devices).

C₂C₁₂ mouse C3H muscle myoblasts (cat. no. 91031101, Sigma-Aldrich) were cultured as previously described [28 (link)]. Proliferating C₂C₁₂ cells were propagated in DMEM (ThermoFisher) supplemented with 5% fetal bovine serum (FBS), 20 mM HEPES, 100 U ml^-1 penicillin, 100 μg ml^-1 streptomycin and 1% L-glutamine in a humidified atmosphere of 95% air/5% CO₂ at 37°C. Differentiation was achieved upon exposure of proliferating C₂C₁₂ cells to a differentiation medium (DM) containing DMEM (25 mM glucose) supplemented with 2% horse serum, 20 mM HEPES, 100 U ml^-1 penicillin, 100 μg ml^-1 streptomycin and 1% L-glutamine for six days. To assess C₂C₁₂ myotube treatment, cells were first synchronized in fresh DM for 24 hours and then treated with the CB₁ receptor agonist ACEA or the CB₁ receptor antagonist AM251 at concentrations of 20, 50, 500, 10³ and/or 5∙10³ nM for 2 hours. After treatment, the cells were first cultured in fresh DM without serum for 2 hours and then incubated in 10 nM insulin for 10 minutes. Finally, 0.2 mg ml^-1 Nitroblue Tetrazolium (NBT) was also added into the medium for a 3-hours incubation at 37°C in order to detect diaphorase/oxidative reaction at an optic density of 560 nm (VersaMax Absorbance Microplate Reader, Molecular Devices).

The MIC assay was performed following the guidelines from the Clinical and Laboratory Standards Institute with slight modifications (16 (link)). Briefly, P. aeruginosa was incubated at 37°C overnight and diluted in cation-adjusted Muller-Hinton broth (CAMHB) to approximately 1 × 10⁶ CFU/mL. Bacteria were incubated with 2-fold diluted carbapenems or peptides in 96-well plates (Greiner Bio-One, Frickenhausen, Germany) at 37°C overnight. After incubation, the final OD₆₀₀ was measured using the VersaMax absorbance microplate reader (Molecular Devices, CA, USA), and the minimal inhibition concentration was determined by the concentration of wells at which no bacterial growth was detected. The assay was performed in triplicate, with each sample performed in triplicate wells.

To cultivate bacteria for growth experiments, V. anguillarum cells grown overnight at 27 °C in LB20 supplemented with the appropriate antibiotics were harvested by centrifugation (9000×g, 2 min), washed twice and resuspended in in NSS. A 200 μl aliquot of the V. anguillarum NSS suspension was transferred into a 96-well plate with a clear flat bottom and the optical density at 600 nm (OD₆₀₀) was read by a VersaMax™ Absorbance Microplate Reader (Molecular Devices). The V. anguillarum NSS suspension was prepared to an OD₆₀₀ of 0.420 (~4 × 10⁷ CFU/ml) and diluted 1:100 into fresh media. Growth was monitored either by measurement of the OD₆₀₀ or by serial dilution and plate counts.