Spectramax i3 spectrophotometer

hAECs were thawed and seeded in T175 flasks overnight at 37 °C and 5% CO₂. The following morning, cells were lifted using 0.05% trypsin and seeded at a density of 0.5 × 10⁶ cells/well in a 24-well plate (BD Falcon). Cells were stimulated with LPS (100 ng/ml) for 24 h and supernatant collected and spun down briefly to remove particulates (500 × g for 5 min). Levels of IL-6 (SKU0029; ELISAkit, Melbourne, VIC, Australia), IL-10 (SKU0007; ELISAkit), IL-37 (ELH-1L1F7; RayBioTech, GA, USA) and IL-1Ra (ab100565; abcam, Cambridge, UK) were measured in duplicate following the manufacturers’ instructions and quantified on a SpectraMax i3 Spectrophotometer (Molecular Devices).

Leptin and adiponectin concentrations were measured at day 9 in fully differentiated SGBS cells. Cells were washed two times with serum-free Basal medium and left untreated or treated with 100 nM insulin. After 6 h of incubation, aliquots of the medium were removed and frozen at −20 °C for leptin or adiponectin measurements respectively.
Leptin and adiponectin secretion were measured using the human leptin or adiponectin DuoSet ELISA Development kits (R&D Systems, Inc., Minneapolis, MN, USA, sensitivity: 0.031 ng/ml for leptin and 0.062 ng/ml for adiponectin respectively). The assays were conducted in 96-well microplates and read out on a SpectraMax i3 spectrophotometer (Molecular Devices, LLC, Sunnyvale, CA, USA). The absorbance value was measured at 450 nm and normalized to the protein concentration measured by the Bio-Rad DC Protein Assay.

The levels of oxidative stress were determined by assessing the extent of lipid peroxidation by measuring the levels of malondialdehyde (MDA) using the thiobarbituric acid reactive substances (TBARS) assay kit (Cayman Chemical, Michigan, USA). The levels of total protein were quantitated by using the Pierce^TM bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific Inc, Minnesota, USA) according to the manufacturer's manual. In brief, the frozen sciatic nerve was homogenized in 250 µL of RIPA buffer with PIC and centrifuged at 1600 g for 15 minutes at 4°C; the supernatant was collected and used for the measurement of the levels of MDA and total protein. For measurement of MDA levels, the supernatant was stimulated by the addition of sodium dodecyl sulfate solution. The reactions were terminated by adding acetic acid solution and thiobarbituric acid reactive substance reagent. After boiling for 1 hour, the samples were cooled on ice for 30 minutes and centrifuged at 1600 g for 10 minutes at 4°C. The samples were collected and were used to measure the MDA levels at 535 nm using Spectra Max i3 spectrophotometer (Molecular Devices LLC., Tokyo, Japan). The levels of MDA were determined using the standard curve and were calculated as nmol/mg total protein content.

Microglia viability and proliferation was assessed using the Promega MTS assay (G5421; Promega), according to the manufacturer’s instructions. Briefly, 5 × 10³ microglia were plated into each well of a 96-well plate (BD Falcon). Primary microglia were stimulated with LPS with or without hAEC-conditioned medium (n = 3 animals for each group). For each treatment and time-point, 20 μl of MTS solution was added, and microglia were incubated for 4 h. Optical density was measured at 490 nm with the SpectraMax i3 Spectrophotometer (Molecular Devices, CA, USA). All samples were run in triplicate and analysed using the built-in SoftMax Pro software suite.

Indole concentrations in the stools were determined using the hydroxylamine indole assay (15 (link)). Briefly, 250 mg of stool was suspended in 750 µl of 70% ethanol, vortexed for 30 s, and incubated in a 70°C water bath for 10 min. The samples were vortexed again for 30 s and centrifuged at 14,000 × g for 20 min at 40°C, and the supernatants were carefully pipetted. For the indole test, 100 µl of the supernatant was added in triplicates to a Costar 96-well plate (Corning, NY) containing 25 µl of NaOH (5.3 M) and 50 µl of 0.3 M hydroxylamine hydrochloride (NH₂OH-HCl) and incubated for 15 min at room temperature. Following the incubation period, 125 µl of H₂SO₄ (2.7 M) was added, thoroughly mixed, and incubated at room temperature for 2 to 30 min. Absorbance at a 530-nm wavelength was measured using a Spectramax I3 spectrophotometer (Molecular Devices, Sunnyvale, CA). The concentration of indole in the sample was determined using a standard curve obtained from known indole concentrations.

To analyze the sensitivity of the Ent. faecalis 14 mutant strains, and other targets to the purified EntDD14, kinetics at 37°C in GM17 medium, supplemented with purified EntDD14 at a final concentration of 10 μg/mL, were performed in a 96-well microplate using a SpectraMax i3 spectrophotometer (Molecular Devices, CA, USA). The cultures were inoculated with the same bacterial charge thus giving the same initial OD₆₀₀ of 0.2. Measurements were taken every 30 min at OD₆₀₀ for 12 H. In parallel, the activity of EntDD14 (60 μg/mL) or serial dilutions of culture supernatant was also tested using the well-diffusion assay and the bacteriocin activity was expressed in AU/mL, which corresponds to the inverse of the last active dilution multiplied by 100 (Batdorj et al., 2006 (link)).