Microtubule seeds were prepared at 10 μM tubulin concentration (30% ATTO-488-labeled or ATTO-565-labeled tubulin and 70 % biotinylated tubulin) in BRB80 supplemented with 0.5 mM GMP-CPP at 37°C for 1 h. The seeds were incubated with 1 μM Taxotere (Sigma) at room temperature for 30 min and were then sedimented by high centrifugation at 30°C and resuspended in BRB80 supplemented with 0.5 mM GMP-CPP and 1 μM Taxotere. Seeds were stored in liquid nitrogen and quickly warmed to 37°C before use.
The PDMS chip was placed on a micropatterned cover glass and fixed on the microscope stage. The chip was perfused with neutravidin (25 μg/ml in BRB80; Pierce), then washed with BRB80, passivated for 20 s with PLL-g-PEG (Pll 20K-G35-PEG2K, Jenkam Technology) at 0.1 mg/ml in 10 mM Hepes (pH = 7.4), and washed again with BRB80. Microtubule seeds were flowed into the chamber at high flow rates perpendicularly to the micropatterned lines to ensure proper orientation of the seeds. Non-attached seeds were washed out immediately using BRB80 supplemented with 1% BSA. Seeds were elongated with a mix containing 14, 18, 20 or 26 μM of tubulin (30% labeled) in BRB80 supplemented with 50 mM NaCl, 50 25 mM NaPi, 1 mM GTP, an oxygen scavenger cocktail (20 mM DTT, 2 mg/ml glucose, 80 μg/ml catalase and 0.67 mg/ml glucose oxidase), 1% BSA, 0.025% methyl cellulose (1500 cp, Sigma) and 0.02% red fluorescent beads (Fluoro-Max polymer spheres, 0.52 μm diameter, Thermo Scientific). The same mix was used to subject microtubules to flow. For the experiments showing tubulin incorporation after bending, a mix was prepared with 100% labeled tubulin for bending the microtubules.
The PDMS chip was placed on a micropatterned cover glass and fixed on the microscope stage. The chip was perfused with neutravidin (25 μg/ml in BRB80; Pierce), then washed with BRB80, passivated for 20 s with PLL-g-PEG (Pll 20K-G35-PEG2K, Jenkam Technology) at 0.1 mg/ml in 10 mM Hepes (pH = 7.4), and washed again with BRB80. Microtubule seeds were flowed into the chamber at high flow rates perpendicularly to the micropatterned lines to ensure proper orientation of the seeds. Non-attached seeds were washed out immediately using BRB80 supplemented with 1% BSA. Seeds were elongated with a mix containing 14, 18, 20 or 26 μM of tubulin (30% labeled) in BRB80 supplemented with 50 mM NaCl, 50 25 mM NaPi, 1 mM GTP, an oxygen scavenger cocktail (20 mM DTT, 2 mg/ml glucose, 80 μg/ml catalase and 0.67 mg/ml glucose oxidase), 1% BSA, 0.025% methyl cellulose (1500 cp, Sigma) and 0.02% red fluorescent beads (Fluoro-Max polymer spheres, 0.52 μm diameter, Thermo Scientific). The same mix was used to subject microtubules to flow. For the experiments showing tubulin incorporation after bending, a mix was prepared with 100% labeled tubulin for bending the microtubules.
