Vw h2ma

To obtain images of mitochondria, cells from sIBM patients were stained with MitoTracker red (a molecular probe, shown in red), and the nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI, shown in blue). The samples were then placed into a confocal microscopy system (Nikon, Tokyo, Japan). Images were acquired and analyzed using NIS-Elements with N-SIM analysis software (Nikon) and ImageJ. Briefly, the MitoTracker red signal in the images was changed to grayscale, and a suitable threshold level that allowed the signal intensity of the mitochondria to be distinguished from the background noise was set in ImageJ software. The lengths of the major and minor axes of the mitochondria were measured using ImageJ. Electron microscopy analysis was performed as previously reported [12 (link), 16 (link)]. For live-cell imaging, culture dishes with myoblasts and fibroblasts from sIBM patients stained with MitoTracker Green were placed into a KEYENCE BZ-X700 All-in-one Fluorescence Microscope (KEYENCE, Osaka, Japan). Images were taken for 5 min and converted to movie files using a BZ-X Analyzer (KEYENCE). The movies were analyzed with the video editing analysis software VW-H2MA (KEYENCE) to evaluate cell migration [17 (link)].

To investigate contractility of the skeletal muscle tissue formed on the flexible substrate, we applied alternative-current (AC) square waves as electrical stimulations, generated using a function generator (Agilent Technologies Inc.) and an amplifier (Mess-Tek Co., Ltd.), to the skeletal muscle tissue via gold electrodes placed in the culture medium. To measure the moving distance of the tip of the flexible substrate, we observed the tip using a microscope (IX71N; Olympus) and quantified the movements of the tip using a motion analyzer (VW-H2MA; Keyence Corp.).
To evaluate the motions of the biohybrid robot in air, we placed it in air by immobilizing wires on a fixed base. In this state, we applied AC square waves to the skeletal muscle tissue via gold electrodes patterned on the flexible substrate. To estimate the moving distance of the reference point, we observed the point using the digital camera with a macro lens and quantified the movements of the tip using the motion analyzer.

We performed real-time imaging of GC cells in 6-well plates. Each well was coated with 200 μl of BD Matrigel (BD Biosciences, California, USA). NUGC3/C5aR cells were plated and allowed to adhere for 12 hours. The 6-well plates were imaged with a KEYENCE BZ-X700 all-in-one fluorescence microscope equipped with a CO₂ and temperature controlled chamber and time-lapse tracking system (KEYENCE, Osaka, Japan). Phase contrast images were taken every 10 minutes for 24 hours and converted to movie files using a BZ-X Analyzer (KEYENCE). We analyzed the movies for cell migration with the video editing analysis software VW-H2MA (KEYENCE) and subsequently processed the tracking data with Microsoft Excel 2010 (Microsoft, Redmond, WA) to create xy coordinate plots and distance measurements.