35 mm glass bottom imaging dish

Cells were seeded (10⁵) on a 35 mm glass bottom imaging dish (MatTek) in normal medium (DMEM/F12, HEPES with 5% FBS, Gibco). The day after, cells were washed twice in PBS before being serum starved in Phenol Red-free medium (DMEM/F12, HEPES, Gibco) for 24 h. Cells were imaged at 37°C with CO₂ on an Olympus/Abbelight SAFe360 system with two Hamamatsu Fusion sCMOS cameras, 488 nm diode laser and an 100× oil immersion objective. The pixel size was 99.7 nm. The cell surface plane was found using automated TIRF angles in the mNeonGreen channel. Abbelight NEO software was used for acquisition of movies, with frames captured every 100 ms for no longer than 1 min per movie and no more than 1 h per dish.

A chloroform solution of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine with 0.1 mole percent 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Avanti Polar Lipids) was dried first under a stream of nitrogen and then under vacuum to remove the solvent. The resulting film was dissolved to 1 mg/mL in PBS pH 7.4 at 37 °C with extensive vortexing and freeze-thawed 7x by cycling between liquid nitrogen and 37 °C baths. The lipid suspension was then extruded 13x through double stacked 100 nm polycarbonate membranes (Whatman) to yield small unilamellar vesicles (SUVs). SUVs prepared in this way were stored at 4 °C and used within 2 weeks. Silicon wafers with ~1900 nm thermal oxide (Addison Engineering) were diced into 1 cm² chips, and the oxide layer thickness for each chip was measured with a FilMetrics F50-EXR. The chips were then cleaned in piranha solution (30 volume % hydrogen peroxide in sulfuric acid), rinsed extensively and stored in deionized water until use. SLBs were formed by incubating the cleaned silicon chips in the SUV solution for 1 hour, and then rinsed with PBS, incubated with 1.75 μM DiIC₁₈ for 30 minutes, and rinsed again with PBS. The samples were then inverted into a 35 mm glass-bottom imaging dish (MatTek) containing PBS and immediately imaged.