CaOx Crystal Aggregation Assay

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.