Stage top incubator

PER2::LUC is a luciferase fusion protein that reports its expression bioluminescently. Time-dependent changes in PER2::LUC expression were measured in real-time via a cooled-CCD camera (imaging) or a photomultiplier (luminometry). Bioluminescence imaging was performed in a previously described custom-made, light-sealed inverted microscope system [26 (link)], which has a simplified optical path optimized for low-light imaging, with a 4× objective lens and a 0.35× relay lens (Olympus). The cultured sample was maintained at 37 °C in a stage-top incubator (Tokai HIT, Shizuoka, Japan), and time-lapse imaged with an Orca R2 cooled-CCD camera (Hamamatsu Photonics, Hamamatsu, Japan) with external water-cooling (20 °C; TGV-10, Taipei, Taiwan). Images were taken under 1 h exposure at 4×4 binning, at a sampling interval of 1 h. For luminometry, an equal number of control and test samples were measured on the same 8-dish wheel in Kronos Dio (ATTO, Tokyo, Japan); light from each dish was measured under 1 min exposure at sampling interval of 10 min, with the nominal temperature of 37 °C and actual temperature maintained between 37.6–37.7 °C, measured with iButton datalogger in the dish (DS1921L, Maxim Integrated, San Jose, CA, USA). Bioluminescence traces were detrended using the HP filter as described previously [23 (link)].

In order to visualize acidic compartments, live cell imaging was performed for 661W cells stained with AO (R&D systems). 661W cells were plated in a multi-well glass bottom dish (Matsunami Glass Industry) at a density of 10,000 cells per well and incubated overnight at 37 °C. The medium was replaced with DMEM containing 1% FBS, and the cells were incubated with either 10 µg/mL MBE, 10 µM D3S5G or 10 µM D3G5G for 1 h at 37 °C, followed by exposure to blue light for 8 h. AO was added to a final concentration of 1 mg/mL, and the cells were incubated for 20 min at 37 °C. Finally, the medium was changed to prewarmed Live Cell Imaging Solution (LCIS, Thermo Fisher Scientific) containing 5.5 mM glucose and 1% FBS, and cells were subjected to live cell imaging using a 40× oil lens on an FV3000 microscope (Olympus Co.) equipped with a stage top incubator (Tokai Hit Co., Shizuoka, Japan). AO green and red fluorescence intensities were measured using ImageJ Ver. 1.53c (National Institutes of Health) for 24 cells each in four independent experiments to obtain the intensity ratio of green vs. red.

Twelve h after co-culturing PTECs with MtDsRed2-MSCs or the addition of DsRed2-Mt, time-lapse observations and imaging of phase contrast and fluorescence were commenced with Axio Observer Z1 (Carl Zeiss) at 37 °C in 5% CO₂ using a stage top incubator (Tokai Hit, Fujinomiya, Japan).