Go microplate reader

Lipid accumulation in the differentiated hBM-MSC adipocytes was observed and evaluated with Oil Red O staining. At the end of the differentiation period (day 12), cell culture wells were washed with PBS, and the adipocytes were fixed on wells by adding 1 mL formalin (neutral buffered, 10%) and incubating for 1 h. After fixation, formalin was aspirated, wells were air-dried, and 0.5% Oil Red O staining solution (wt/v, in 3 parts isopropanol and 2 parts triple-distilled water) was added to each well for staining. Staining lasted for 1 h at room temperature, after which wells were subsequentially aspirated, washed with PBS twice, and photographed. Lipid accumulation was also quantified by measuring the retained Oil Red O stain by intracellular lipid deposits. The stain was eluted from cells in each well by adding 1 mL of 10% isopropanol. Plates were then let rest at room temperature for the stain to be removed fully. The optical density of each well was then measured at 500 nm with a MultiSkan GO microplate reader. Obtained absorbance values were used to evaluate the lipid accumulation by plotting it as a relative percentage of the differentiated untreated hBM-MSC adipocytes.

The peptidoglycan integrity was assessed by measuring susceptibility to lysozyme‐mediated bacterial lysis.^[63] After incubation overnight, log‐phase cultures were prepared in fresh nutrient broth in the absence or presence of KTA (0.625 µmol L⁻¹, 1/4 × MIC) or KTR (1.18 µmol L⁻¹, 1/4 × MIC) and then incubated for 2 h. Cells were resuspended in 10 mm HEPES (pH 7.5) and adjusted to an OD_{600 nm} of 2.0. Untreated and treated cell suspensions were mixed with an equal volume of HEPES buffer and 10 mm EDTA. After the addition of 1 mg mL⁻¹ lysozyme, cell lysis was assessed over 1 h at 25 °C by monitoring OD_{600 nm} every 2 min using a Multiskan GO microplate reader.