12 well glass bottom plate

HeLa‐H2B/tub cells 30 expressing H2B‐mCherry and EGFP‐α‐tubulin were kindly provided by Jan Ellenberg (EMBL Heidelberg, Germany) and used for time‐lapse microscopy as described previously 67. Briefly, cells were plated on μ‐slide 8‐well ibiTreat chambers (Ibidi) and imaged at 5‐min intervals for 48 h on a Nikon BioStation IM‐Q Time Lapse Imaging System using a 20×/0.8 NA air objective, a 1.3 megapixel cooled monochrome camera (Nikon), and Nikon software for image acquisition. Alternatively, HeLa‐H2B/tub cells were imaged at 15‐min intervals on a DeltaVision deconvolution microscope using a 20×/0.75 air objective.
HeLa_dox‐YFP‐TIARr cells were plated on a 12‐well glass bottom plate (Cellvis) and imaged at 15‐min intervals for 12 h on a Nikon Ti‐E microscope with an integrated perfect focus system using a 40x/0.95 NA air objective, a 4.2 megapixel cooled monochrome sCMOS pco.pge camera, and NIS elements JOBS software for image acquisition. All cells were kept in 10% FBS/DMEM at 37°C and 5% CO₂ during imaging. Image processing was performed using ImageJ software (NIH, http://rsbweb.nih.gov/ij/).

The formation of TNTs was monitored in 2D co-culture using time-lapse confocal microscopy. Briefly, a 12-well glass bottom plate (Cellvis) was plasma treated for 3 min on high using an expanded plasma cleaner (Harrick Plasma) and UV treated for 1 h before plating 15,000 MDA-MB-231 cells in complete growth medium. Cells were allowed to adhere for ∼72 h before time-lapse imaging. At ∼24 h before imaging, MitoTracker Green (Invitrogen), reconstituted in DMSO, was diluted in low-glucose DMEM to a staining concentration of 12.5 nM. A T-25 flask of CAF01-hTERTs were rinsed twice with 5 ml of 1× PBS, incubated in the MitoTracker Green staining solution for 30 min, before being rinsed twice with 5 ml of 1X PBS, and cultured in CAF complete growth medium. At 24 h post staining, 20,000 MitoTracker Green-labeled CAF01-hTERTs were seeded onto the 12-well glass bottom plate wells containing MDA-MB-231s in MDA-MB-231 complete growth medium. The plate was equilibrated for 30 min in the LSM800 environment chamber before selecting positions and beginning imaging. The plate was imaged every 8.7 min for 6 h using a 20× NA 0.8 dry lens. 3D co-culture spheroids were imaged with a 20× NA 0.8 dry lens, following the procedure of imaging migration in spheroids. The number of cells per field containing transferred mitochondria were counted manually using ImageJ from both 2D and 3D conditions.

In the TNF (20 ng/ml) challenged cultures, the apical surfaces were washed with pre-warmed PBS the day before addition of 50 μL of CountBright Absolute Counting beads (Invitrogen, Carlsbad, CA, USA) diluted 1:10 000 in PBS with Mg²⁺ and Ca²⁺. Inserts were transferred to a 12-well glass bottom plate (Cellvis, Mountain View, USA) and monitored in a Zeiss LSM 880 (Carl Zeiss AG, Oberkochen, Germany) at 37°C. Three regions in each well of each condition in three COPD donors were filmed and analyzed. Beads were tracked in the ImageJ software with the Fiji plugin Tracking [55 (link)] and the mean velocity of each bead was plotted.