Secure seal hybridization chamber

Cover glasses intended for photobleaching experiments via TIRFM and not ZMW fabrication were cleaned by successive sonication for 60 minutes in 2% Hellmanex (Hellma), HPLC-grade ethanol (Millipore Sigma) and 1 M KOH, with deionized water rinses between solution exchanges. Both cover glasses and ZMW chips were additionally plasma cleaned for 5 minutes prior to surface functionalization. For ZMWs, the Al layer was passivated by incubation in 2% poly(vinylphonic acid) (PVPA) (Polysciences) for 3 minutes 90°C, followed by rinsing with Milli-Q ultrapure water and drying with Ar gas^{38 (link)}. A silicone-gasketed chamber was attached to each substrate to hold small volumes and reduce evaporation (SecureSeal Hybridization Chambers, Grace Bio-Labs,). Both cover glasses and ZMW chips were silanized overnight in 2 mg/mL biotin-PEG-silane (MW = 3400 g mol⁻¹) and 10 mg/mL mPEG-silane (MW = 2000 g mol⁻¹) (Laysan Bio Inc.) in HPLC-grade ethanol (Millipore Sigma) with 5% glacial acetic acid. Samples were rinsed thoroughly with HPLC-grade ethanol, Milli-Q ultrapure water, and dried with Ar gas. Samples were additionally incubated with 10 mg/ml bovine serum albumin (BSA) in tris buffered saline (TBS: 300 mM NaCl, 20 mM Tris HCl, pH = 7.9) for 30 minutes to ensure robust passivation.

The human bladder carcinoma cell line T24 (DSMZ # ACC 310, Leibniz Institute DSMZ) stably transfected with mGFP-GPI(DAF) was cultured in RPMI 1640 medium containing 10% fetal calf serum. HeLa cells were cultured in Dulbecco’s Modified Eagle Medium/HAMS F-12 Medium 1/1 supplemented with 2mM L-glutamine and 10% fetal calf serum. Cells were grown in a humidified atmosphere at 37°C and 5% CO₂. HeLa cells were transiently transfected with mGFP-GPI(hFR) using TurboFect Transfection Reagent (Thermo Scientific) 24 hours prior to experiments according to the manufacturer’s protocol. For micropatterning experiments, cells were grown to 80% confluency and harvested by treatment with accutase. Cells were seeded onto the micropatterned surfaces in Secure-Seal hybridization chambers (Grace Biolabs) and grown for two hours before measurements. For cholesterol depletion, patterned cells were incubated in the presence of 1 U/mL cholesterol oxidase in HBSS at 37°C for 20 min. The extent of cholesterol depletion was determined using the Amplex Red Cholesterol Assay Kit (Invitrogen) following the manufacturer’s instructions.