Nis element d v 4

COM crystallization assay was performed according to a protocol established previously18 (link)40 (link). Briefly, 0.5 ml of 10 mM calcium chloride (Sigma-Aldrich; St. Louis, MO) in basic buffer containing 10 mM Tris and 90 mM NaCl (VWR; Leuven, Belgium) was added into 24-well polystyrene plate (Corning Inc.; Corning, NY) in the absence or presence of purified caffeine (Sigma) with various concentrations of 1 μM, 10 μM, 100 μM, 1 mM, and 10 mM. For the control, an equal volume of the basic buffer was added instead of caffeine. Thereafter, 0.5 ml of 1 mM sodium oxalate (Sigma-Aldrich) in basic buffer was added into each well of the mixture. After 60-min incubation at room temperature (RT) (set at 25 °C), the crystal images were taken using an inverted light microscope (Nikon eclipse Ti; Nikon; Tokyo, Japan). Crystal size was then analyzed using crystal area measured by NIS-element D V.4.11 software (Nikon) from at least 100 individual crystals in each well. Crystal number in each well was counted from 15 high-power fields (HPFs). Crystal mass was calculated using the formula:

COM crystals were prepared in 24-well polystyrene plate by mixing 10 mM calcium chloride with 1 mM sodium oxalate in basic buffer to make their final concentrations of 5 mM and 0.5 mM, respectively. After 60-min incubation at RT, caffeine with various doses (1 μM, 10 μM, 100 μM, 1 mM, and 10 mM) was added to the crystal mixture and the crystals were immediately imaged (set as T₀) under an inverted light microscope (Nikon eclipse Ti). After further 60-min incubation at RT, crystal images were then again captured (set as T₆₀) under an inverted light microscope (Nikon eclipse Ti). The crystal sizes at T₀ and T₆₀ were then analyzed from at least 100 individual crystals per well using NIS-element D V.4.11 software (Nikon) and crystal growth was calculated from Δ crystal area using the formula: