Plasma cleaner

On the day of plating, planar multi-electrode arrays (59 titanium nitride electrodes, 30 μm-diameter, 200 μm-spacing, internal reference electrode; MultiChannel Systems, Fig. S1) were sterilized in a plasma cleaner (Diener Electronic). The central-most portion of the culture compartment was treated with an 8 μl drop of poly-d-lysine (Sigma) (0.5 mg/ml), washed with 8 μl sterile water, then coated with a 4 μl drop of ice-cold 1 mg/ml laminin (Invitrogen). 30 μl of full Neurobasal medium was dispensed round the perimeter of the culture compartment of the MEA prior to the laminin coating step. MEAs were fitted with a sterile, gas-permeable/water vapour-impermeable lid (Potter and DeMarse, 2001 (link)) and placed in an incubator (37 °C; 5%CO₂/95% air; humidified) until required for plating.

The MEA2100 system from Multi Channel Systems (Reutlingen, Germany) was used to perform electrophysiological recordings (chapter Multielectrode Array Recordings) of murine retinae. It comprised a head stage with an integrated preamplifier, which was used for recording as well as for stimulation, and an interface board that served as digital/analog converter transmitting data in real time. A MEA was placed in the head stage that was connected to the interface board, which was connected to a personal computer (PC). The setup was placed on an air-suspended table (Ametek, Berwyn, PA, United States) in a faraday cage (Ametek) to minimize noise caused by vibrations and electronic devices.
60MEA200/30iR-Ti-pr-T type MEAs (Multi Channel Systems) were used. The MEAs possess 60 titanium nitride (TiN) electrodes arranged in a square field of 8 × 8 electrodes with the four electrodes at the corners being spared out and one electrode serving as reference, resulting in 59 electrodes for recording and stimulation. The electrodes were 30 μm in diameter and positioned with a distance of 200 μm to each other. All MEAs had a plastic ring around the electrode field with an inner diameter of 26 mm and a thread on the inside. Before every experiment, MEAs were hydrophilized with oxygen plasma for 2 min at 0.5 mbar in a plasma cleaner (Diener Electronic, Ebhausen, Germany).

HS-AFM movies were acquired with an HS-AFM (SS-NEX, Research Institute of Biomolecule Metrology, Tsukuba, Japan) equipped with a superluminescent diode (wavelength, 750 nm; EXS 7505-B001, Exalos, Schlieren, Switzerland) and a digital high-speed lock-in Amplifier (Hinstra, Transcommers, Budapest, Hungary) detailed in Colom et al., 2013 (link) following the protocol detailed in Zuttion et al., 2018 (link). Scanning was performed using USC-1.2 cantilevers featuring an electron beam deposition tip (NanoWorld, Neuchâtel, Switzerland) with a nominal spring constant k = 0.15 N/m, resonance frequency f(r) = 600 kHz, and quality factor Qc ≈ 2 under liquid conditions. For high-resolution imaging, the electron beam deposition tip was sharpened by helium plasma etching using a plasma cleaner (Diener Electronic, Ebhausen, Germany). Images were acquired in amplitude modulation mode at the minimal possible applied force that enables good quality of imaging under optical feedback parameters.

Sheep blood (Oxoid, Thermo scientific, Waltham, MA, USA) was removed by washing with PBS pH 7.4 three times. The Sheep blood was gentle mixed with PBS in a ratio of 1:7. Then, the erythrocytes were collected by centrifugation at 3000× g and 4 °C for 5 min. The erythrocyte pellet was kept and resuspended in PBS pH 7.4, 2% (V/V), as a working solution.
The erythrocyte membrane was prepared on a poly-lysine coated glass. The round-glass cover slips were cleaned as follows: soaking in 1.0 M hydrochloric acid for 2 h, rinsing thoroughly with ultrapure water (MilliQ, Merck, Darmstadt, Germany), sonication in 70% (v/v) ethanol for 10 min, and final treatment with plasma cleaner (Diener electronic, Ebhausen, Germany). Prior cell attachment, the glass cover slips were coated with 30–70 kDa poly L-lysine (Sigma, Darmstadt, Germany). The glass slips were immersed in a 0.1 mg/mL lysine solution (in PBS) for 30 min at room temperature. The excess of lysine was removed by buffer rinsing. After that, the erythrocytes were attached on the glass surface by incubation over the surface for 30 min at room temperature. The unbound erythrocytes were removed and the attached cells were opened under shear flow by using a low content salt solution (1/3 dilution PBS; 45.7 mM NaCl, 0.9 mM KCl and 3.3 mM phosphate). Finally, the cell membrane was rinsed with PBS pH 7.4.

The AFM cantilevers with a nominal spring constant of 0.24 N/m (DNP-S10, Bruker, Billerica, MA, USA) and the silica substrate were mounted inside a closed fluid cell with an O-ring. The 1 cm × 1 cm silica wafers (IMEC, Leuven, Belgium) were cleaned before using with the following procedure: sonication in 2% (w/w) SDS solution for 15 min, rinsing with ultrapure water, and drying under nitrogen stream. Finally, the substrates were treated by plasma cleaner (Diener electronic, Ebhausen, Germany). The lipid bilayers were formed by means of lipid vesicle fusion. 0.1 mg/mL of lipid vesicle solutions were incubated over the silica surface for at least 10 min and then the vesicle excess was rinsed from the chamber. Afterwards, the two Cyt2Aa2 proteins, wild type (WT) and the T144A mutant (25 µg/mL or 1.0 µM), were incubated with the lipid bilayers or with supported erythrocyte membrane for the desired experimental time. The surface topography was imaged in tapping mode with a JV-scanner controlled by a NanoScope V controller (Bruker, Billerica, MA, USA) at a scan rate of 1–2 Hz. The images were processed and analyzed with the Nanoscope program. The experiments were carried out at room temperature (298 K).