Interchangeable dish

Cells were imaged on 30-mm no. 1.5 coverslips in an interchangeable dish (Bioptechs). Coverslips were plasma-cleaned for 2 min with H₂/O₂. Coverslips were coated with 50 µg/ml Poly-D-Lysine (Sigma-Aldrich) for 20 min at room temperature. Cells were seeded at 50,000 cells per coverslip. Cell dishes were maintained at 34°C in phenol red–free DMEM supplemented with 10% FBS and 20 mM Hepes (pH 7.3) while imaging. Images were acquired in TIRF mode every 1 s (1 FPS) for 2 min. Cell areas were divided by 15° radial lines centered at the observed MTOC. Cell centers were defined as the area within the inner half region of a cell determined by connecting the midpoints of all radial lines. Cell edges were defined as the outermost one quarter of a cell. Kymographs were generated by plotting the track of MT plus-tip marker fluorescence, mCherry-MACF43, as a function of time. MT plus-end growth rates were determined by measuring the displacement of MT plus-tip tracker over time. At least 150 tracks of mCherry-MACF43 were quantified for each assay condition (n ≥ 150).

Cells were imaged on 30 mm #1.5 coverslips in an interchangeable dish (Bioptechs). Coverslips were plasma cleaned for 4 min using Ar/O₂ and 2 min with H₂/O₂. Coverslips were coated with 50 μg/ml poly-d-lysine for 20 min at room temperature and 10 μg/ml fibronectin in phosphate-buffered saline (PBS) for 1 h at 37°C and blocked with 1% bovine serum albumin (BSA) in PBS for 1 h at 37°C. Cells were seeded at 100,000 cells per coverslip. Live-cell microscopy was performed on a Nikon Ti-E microscope with a 100× TIRF objective (NA = 1.49), an Andor Zyla 4.2 sCMOS camera, and Nikon Elements software. The microscope was equipped with a perfect focus system and automated TIRF angle motor. Cell dishes were maintained at 37°C in a heated chamber, and the objective was warmed using a heating collar (Warner Instruments). Cells were cultured in phenol red-free DMEM supplemented with 10% FBS and 20 mM HEPES (pH 7.3). Excitation was performed with a 405, 488, or 561 nm laser, as appropriate. For live-cell time-lapse movies, the Zyla 4.2 camera was binned at 2 × 2 pixels and acquisitions were performed with 400 ms integration times. Images were acquired in epifluorescence and TIRF mode every 10 s or in TIRF mode every 2 s.