Perfection v200

The excreta of adult flies are small, almost transparent droplets or semi-solid deposits. To aid their visualisation, the inexpensive, non-toxic dye Bromophenol blue sodium salt was added to the flies’ diet to a concentration of 5 g/L (Pulver et al., 2011 ). At this concentration, the dye does not affect the palatability of the food tested by two-choice CAFÉ assay and no developmental delay is observed if fed during the larval stage (data not shown). The dye is not altered by heat and can be added to liquid and solid diets. Its hue is however sensitive to pH, shifting from blue at neutral and basic pH to yellow and orange at acidic pH. Because of this, care should be taken in adjusting the pH of the prepared food to equal values across different conditions studied.
To obtain a digital image of fly excreta, groups of 6–8 flies were allowed to excrete onto flat clear plastic surfaces, in this case the lid Petri dishes, for about 60 h. The dye-laced food was offered ad libitum as a wedge of solid food placed on the dish (Pulver et al., 2011 ). At the end of the experiment, the flies and food were removed from the plate and a high-resolution image of the deposits left on the plastic surface was acquired with a transparency scanner (Epson Perfection V200). Image cropping and preparation were performed in Adobe Photoshop CS4.

UMR-106 cells seeded and incubated for 7 days in a six-well plate in the presence and absence of MSM (20 mM) were used to determine the effect of MSM on matrix mineralization. Cells without any MSM but grown in the OM were used as controls. Alizarin red S (ARS) is used to stain cells after washing with phosphate-buffered saline (PBS) three times. Absolute ethanol was used to fix the cells for 30 min at room temperature. After ethanol aspiration, 2% ARS solution was added to each well and processed as described previously (Aljohani et al., 2019 (link)). For Von Kossa staining, cells were washed with PBS three times and fixed with 10% paraformaldehyde for 10 min at room temperature. After the aspiration of fixative and washing with PBS, a 5% silver nitrate solution was used as described previously (Aljohani et al., 2019 (link)). Scanning the culture plates stained for ARS and Von Kossa was done in the scanner (EPSON Perfection V200). Nikon Eclipse TE 2000-inverted light microscope were used to obtain magnified images (10× objective).