Crystallization assay was performed following protocol described in previous studies [18] (link), [19] (link). In brief, 500 μl of 10 mM CaCl2·2 H2O in the crystallization buffer was put into each well of 24-well plate (Corning Inc.; Corning, NY). Then, 4 μl of 1 mg/ml recombinant human CNB, 1 mg/ml TUPs, 1 mg/ml lysozyme (negative control), or crystallization buffer without any protein added (blank control) was added, followed by 500 μl of 1 mM Na2C2O4. The mixture was incubated at 25 °C for 60 min, and the resulting COM crystals were imaged using an inverted light microscope (Eclipse Ti-S; Nikon; Tokyo, Japan). Crystal size and number were measured from at least 100 crystals in 10 random fields per sample using NIS-element D software version 4.11 (Nikon). Crystal abundance was then calculated.
Nis element d software version 4
Manufactured by Nikon
NIS-element D software version 4.11 is a digital imaging and analysis software designed for microscopy applications. It provides a comprehensive suite of tools for image acquisition, processing, and analysis. The software supports a wide range of Nikon microscopes and cameras, enabling users to capture, manage, and analyze high-quality images effectively.
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Lab products found in correlation
3 protocols using nis element d software version 4
1
Calcium Oxalate Crystal Formation Assay
2
CaOx Crystal Growth Assay Protocol
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CaOx crystal growth assay was performed as previously described (Thongboonkerd et al., 2008 (link); Peerapen and Thongboonkerd, 2016 (link)). Briefly, CaOx crystal seeds were generated by adding 500 μl of 1 mM Na2C2O4 (in the basic buffer) into each well of a 24 well-plate containing 500 μl of 10 mM CaCl2 (in the basic buffer) and incubated at 25°C for 60 min. Thereafter, each bacterial component derived from approximately 4 × 107 bacteria and finally resuspended in 4 μl basic buffer was added into each well and this time-point was defined as T0. The mixture was further incubated for 60 min (T60). At T0 and T60, crystal images were captured randomly from at least 15 HPFs under Nikon Eclipse Ti-S inverted phase-contrast light microscope (Nikon). Crystal sizes at T0 and T60 were measured using NIS Element D software version 4.11 (Nikon), whereas crystal growth (represented by Δ Crystal size) was calculated from at least 100 crystals in 15 HPFs in each biological replicate using the following formulas:
(Where T was the Δ Crystal size of the tested sample and B was the Δ Crystal size of the blank control.)
(Where T was the Δ Crystal size of the tested sample and B was the Δ Crystal size of the blank control.)
3
Calcium Oxalate Crystal Growth Assay
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Crystal growth assay was performed following protocol described in previous studies [20] (link), [21] (link). In brief, 500 μl of 10 mM CaCl2·2 H2O in the crystallization buffer was put into each well of 24-well plate (Corning Inc.), followed by 500 μl of 1 mM Na2C2O4. The mixture was incubated at 25 °C for 60 min. At this time-point (T0), 4 μl of 1 mg/ml recombinant human CNB, 1 mg/ml TUPs, 1 mg/ml lysozyme (negative control), or crystallization buffer without any protein added (blank control) was added, followed by another incubation at 25 °C for 60 min (T60). At T0 and T60, COM crystals were imaged using an inverted light microscope (Eclipse Ti-S). Crystal size was measured from at least 100 crystals in 10 random fields per sample using NIS-element D software version 4.11 (Nikon). Δ Area and crystal inhibitory activity were then calculated.
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