Eclipse ti s inverted phase contrast light microscope

CaOx crystal aggregation assay was performed as previously described (Chaiyarit and Thongboonkerd, 2017 (link); Khamchun et al., 2019 (link)). Briefly, individual CaOx crystals were generated as aforementioned but with a larger volume in a 50-ml conical tube (Corning Inc., NY, United States) and then harvested by centrifugation at 2,000 × g for 5 min. The supernatant was discarded, whereas CaOx crystals were washed three times with methanol. After another centrifugation at 2,000 × g for 5 min, methanol was discarded and the crystals were air-dried overnight at 25°C. CaOx crystals (1,000 μg dry weight) were resuspended in 1 ml of the basic buffer in each well of the 6-well plate (Corning Inc., NY, United States). Thereafter, each bacterial component derived from approximately 4 × 10⁷ bacteria and finally resuspended in 4 μl basic buffer was added into each well and then incubated in a shaking incubator at 150 rpm and 25°C for 1 h. Thereafter, formation of CaOx crystal aggregate (defined as an assembly of three or more individual COM crystals that tightly joined together) was observed and imaged under Nikon Eclipse Ti-S inverted phase-contrast light microscope (Nikon). Number of COM crystal aggregates was counted from at least 15 randomized HPFs in each biological replicate.

The floating cells were used for evaluation of cell tolerance to the AU or native urine. The cells (approximately 7.5×10⁴ cells) were seeded in each well of the 24-well plate (Corning Costar; Cambridge, MA) with 1 ml of the complete (serum-containing) culture medium. After incubation in a humidified incubator containing 5% CO₂ at 37°C for 24 h, the cells were washed with PBS and trypsinized with 0.1% trypsin in 2.5 mM EDTA/PBS. Thereafter, the cell suspension was centrifuged at 300 ×g and 4°C for 3 min and the supernatant containing trypsin was removed. The cells were then resuspended in 1 ml of the complete (serum-containing) medium, native urine, or AU-Siriraj with 0-10% FBS supplement. Cell morphology was monitored every 10 min under the Nikon Eclipse Ti-S inverted phase-contrast light microscope (Nikon; Tokyo, Japan). Time to deformation (T_D) was recorded for each sample.