Preparation of CaOx crystals and exposure of MDCK cells to the crystals

CaOx crystals were prepared in an artificial urine as previously described³³. Briefly, 125 ml of 25.08 mM CaCl₂.2H₂O was added into 250 ml of a buffer containing 19.26 mM tri-sodium citrate dihydrate (C₆H₅Na₃O₇.2H₂O), 23.1 mM magnesium sulfate heptahydrate (MgSO₄.7H₂O) and 127.4 mM potassium chloride (KCl). The pH of the solution was adjusted to 6.5 using HCl. The solution was then incubated at RT for 15 min. Thereafter, 125 ml of 6.4 mM sodium oxalate (Na₂C₂O₄) was added under a continuous stirring. The solution was incubated further at RT for 15 min. CaOx crystals were then harvested by centrifugation at 2,000 × g for 5 min. Supernatant was discarded and the crystals were resuspended in methanol. After another centrifugation at 2,000 × g for 5 min, methanol was discarded and the crystals were air-dried. After crystal generation and harvesting, the crystals were decontaminated with UV irradiation for 30 min. They were then added to a complete MEM medium (GIBCO, Invitrogen Corporation) to achieve a final concentration of 1,000 μg of crystals/ml of medium. MDCK cells were cultivated in complete MEM medium without or with crystals for 48 h in a humidified incubator at 37 °C with 5% CO₂.