Visualizing Mitochondrial Transfer via TnTs

Time-lapse imaging experiments were performed on Perkin Elmer UltraView ERS spinning-disk confocal microscope or Zeiss LSM 5Live line-scanning confocal microscope. Both microscopes were enclosed in environmental chambers that were maintained at 37°C with 5% CO₂ level. Viable Staining of Cell Lines for time-lapse imaging was performed as we have described previously (Lou et al., 2012b (link)). Briefly, in order to assess the ability of mitochondria to be transmitted between mesothelioma cells via TnTs, we used MitoTracker Red to stain MSTO-211H cells which were then cultured in hyperglycemic, low-serum (“TnT”) medium. The cells were cultured in clear-bottomed delta-T culture dishes (Bioptechs Inc., Butler, PA). MitoTracker Red CMX Ros (Invitrogen, M-7512, 50 μg/vial) was used at 500 nM to stain mitochondria, per manufacturer's protocols. Stained cells were re-suspended and added to a non-confluent culture of adherent, unstained MSTO-211H cells grown in another dish. Incubation was performed in high glucose medium for 5 h to stimulate formation of TnTs prior to imaging.