Metamorph nx software

EF-induced cell migration experiments were performed as described previously [34 (link)]. Briefly, direct current was applied through agar-salt bridges via Ag/AgCl electrodes in Steinberg’s solution (consisting of 58 mM NaCl, 0.67 mM KCl and 0.44 mM Ca(NO₃)₂, 1.3 mM MgSO₄ and 4.6 mM Tris base, pH 7.4) to pooled medium on either side of the galvanotaxis chamber. Cells were exposed to 0–100 mV/mm direct current EF for 1 h. Cell behavior was observed with a Carl Zeiss Observer Z1 inverted microscope with a Photometrics QuantEM EMCCD camera (Photometrics Inc., Tucson, AZ, USA) and MetaMorph NX software (Molecular Devices, Sunnyvale, CA, USA), and serial time-lapse images were captured. Cell migration was analyzed to determine directedness (cos θ) and track speed by using ImageJ software (NIH, Bethesda, MA, USA) with MTrackJ and Chemotaxis tool plugins. Briefly, trajectories of cells were pooled to make composite graphs. The directedness of migration was assessed as cos θ, where θ is the angle between the EF vector and a straight line connecting start and end positions of a cell. A cell moving directly to cathode would have a directedness of 1; a cell moving directly to the anode would have a directedness of −1. A value close to 0 represents random cell movement. Speed is the total length travelled by the cell divided by time.

The area of MOVAS cells was evaluated after 24 and 48 h exposure to UA (6–12 mg/dL) and compared with that of CTR. In brief, cells grown on chamber slides (EZ chamber slides; Merck Group) were fixed with 2% paraformaldehyde, stained with Hematoxylin–Eosin and photographed with a Leica Microsystems microscope (GmbH Wetzlar, Germany) (40× magnification). Images were then analyzed using MetaMorph^® NX software (Molecular Devices): the areas of randomly selected single cells were defined using the cursor and automatically estimated. The median values were calculated upon measurement of 100 cells/condition.

DMGT and CF41.Mg cells were seeded onto coverslips placed in a 12-well plate and grown to 50–70% confluency. Cells were fixed with 4% paraformaldehyde containing 2% sucrose in 1× PBS at RT for 20 min and then permeabilized with 0.1% Triton X-100 for 3 min and blocked with BlockPRO^TM (Visual protein, Taipei, Taiwan) for 30 min. Cells were incubated with the AGR2 antibody (MA5-16244, Invitrogen, Thermo Fisher Scientific) at 1:100 dilution for 90 min at RT, followed by staining with secondary Alexa Fluor 488-conjugated goat-anti-mouse IgG (Molecular Probe, Thermo Fisher Scientific) at 1:200 dilution for 45 min. The nuclei were co-stained with 4′,6-diamidino-2-phenylindole (DAPI) at 0.1 µg/mL in 1× PBS. Coverslips were washed with 1× PBS between steps and finally mounted with Fluoro-Gel (Electron Microscopy Science, USA) on slides. Results were observed by using Leica DMI3000 Inverted Microscope (Leica, Wetzlar, Germany) equipped with a Zyla 5.5 mega pixel sCMOS camera (Andor Technology, Belfas, Ireland) and processed with MetaMorph^® NX Software (Molecular Devices, San Jose, CA, USA).