50 ml conical tube

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.

Phage lysates were purified using cesium chloride gradient purification as described by ref. ^{69 (link)}. Briefly, 0.5–1 L of the crude lysates were treated with 5 mg/mL each of DNase I and RNAse (Roche) for 1 h at room temperature. The treated lysates were then mixed with NaCl to 1 M and solid PEG-8000 (Fischer) at 10% wt/vol and incubated at 4 °C overnight to precipitate the phage. The next day, the precipitation mixture was centrifuged at 3000 × g at 4 °C for 20 min and supernatant was discarded. The phage pellets were allowed to dry for 5–10 min before resuspension in 2–3 mL of SM buffer. Chloroform was then added at 1:3 of total volume and mixed for 2 min to remove residual PEG. The mixture was then centrifuged for 10 min at 12,000 × g at 4 °C and then the aqueous top layer was transferred to a 50 mL conical tube (Corning Falcon). SM buffer was added to bring the total volume up to 4.5 mL and mixed with 2.25 g of Cesium chloride (CsCl). CsCl density gradients were set up in a Beckman ultra-clear ultracentrifuge tube using 1.7, 1.5, and 1.45 g/mL layers, each at 2 mL, with the phage mixture added on top. The gradient was centrifuged at 60,000 × g at 4 °C for 3 h to produce a visible phage band which was extracted using a sterile 23 G needle. Any remaining CsCl was removed by dialysis using a Slide-a-Lyzer 10,000 MWCO dialysis cassette (Thermo Scientific) in SM buffer.