Fluoroskan microplate fluorometer

The drug susceptibility of M. tuberculosis strains was determined using the resazurin microtiter assay (REMA), as previously described (Palomino et al., 2002 ). Briefly, log-phase bacterial cultures were diluted to a theoretical OD₆₀₀ = 0.0005 and grown in a 96-well black plate (Fluoronunc, Thermo Fisher) in the presence of serial compound dilution. A growth control containing no compound and a sterile control without inoculum were also included. After 7 days of incubation at 37°C, 10 μl of resazurin (0.025% w/v) were added and fluorescence was measured after 24 h further incubation using a Fluoroskan^TM Microplate Fluorometer (Thermo Fisher Scientific; excitation = 544 nm, emission = 590 nm). Bacterial viability was calculated as a percentage of resazurin turnover in the absence of compound.

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.

To investigate intestinal permeability in vivo, we used FITC-dextran (MW: 3,000–5,000, Shanghai Aladdin Biochemical Technology Co., Ltd, China) as a tracer. In brief, after fasting for 12 h, mice were administered with FITC-dextran (44 mg/100 g body weight). 4 h later, blood was collected upon euthanasia and allowed to clot for 1 h. After centrifugation (3,000 rpm, 15 min), serum was collected and diluted with the same volume of PBS. Finally, the FITC-dextran concentration was determined by spectrophotofluorometry (Fluoroskan Microplate Fluorometer, Thermo Fisher Scientific) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. We also determined the serum levels of lipopolysaccharide (LPS) and diamine oxidase (DAO) with ELISA kits (Yingxin laboratory Co., Ltd, Shanghai, China) in accordance with the manufacturer’s instructions (n = 5–6 per group).