Skanit re software

After 3 months of HFD, an IPGTT or OGTT were performed. Briefly, for the IPGTT, 6 h fasted mice were injected with glucose (1 g/kg) into the peritoneal cavity, as previously described [22 (link)]. An OGTT was performed via oral administration of glucose (1.5 mg/g) following a 6 h fast. Blood glucose levels were measured 30 min before glucose administration and at 0, 15, 30, 60, 90 and 120 min following glucose challenge.
For both IPGTT and OGTT, the glycaemic index was calculated as the sum of the blood glucose values (mmol/l) divided by the total time of the curve in min to present value in mmol/l × min, or additionally multiplied by 1000 to give value in μmol/l × min.
Liver triacylglycerol content was measured by a colorimetric assay using free glycerol and triacylglycerol reagents (Sigma Aldrich, St Louis, MO, USA) and the plate was read using the Multiskan Spectrum plate reader and the SkanIt RE software (both Thermo Labsystems, Beverly, MA, USA).

For tests of growth kinetics, liquid media were inoculated with strains propagated on plates. Overnight cultures were diluted 1:100 in a fresh medium and then incubated at 37 °C whilst shaken. When the experiment was performed in M9 medium, overnight cells grown in LB were washed twice with M9 medium and then used for inoculation. Bacterial growth was monitored by the measurement of optical density at 600 nm (OD₆₀₀) in a spectrophotometer or with the use of Varioskan Lux Multimode Microplate Reader and SkanIt RE software (Thermo Fisher Scientific, Waltham, MA, USA) in the case of growth in 96-well plates.

Confluent myotubes were treated with drugs of interest for up to 24 h in differentiation medium prior to resazurin viability assay (Sigma Aldrich, St. Louis, MO, USA) measurements. The resazurin assay solution was prepared at a 1:10 dilution of resazurin in DMEM differentiation medium. Once the dye was added the plates were shielded from light and stored at 37 ${}^{\circ }$ C with 5% CO₂ for two hours. Following the incubation period, the supernatant was transferred to an opaque 96 well plate for fluorometric reading (at 560 nm; Varioskan Flash plate reader) using SkanIt RE software (Thermo Fisher, Waltham, MA, USA). Plates containing myotubes were rinsed with ice cold PBS and fixed with 100% methanol, followed by Diff-Quick staining (Histolabs, Kew East, Australia). Plates were left to dry overnight prior to imaging with an Olympus IX81 microscope (Olympus, Tokyo, Japan) to observe morphological changes.