Zetasizer nano series zs instrument

For bulk nanoparticle size analysis a Malvern Zetasizer Nano series ZS instrument was used to carry out dynamic light scattering (DLS). Measurements were taken directly after the sample suspensions were prepared as described above. A total of at least 3 measurements obtained from an average of 10 runs each were collected and averaged for size analysis. Refractive indices of 1.45 (Lee et al., 2007) and 3.00 (Lide, 2000) were used for the SiO₂ and Fe_xO_y nanoparticles respectively, and 1.33 (Hale & Querry, 1973) and 1.34 (Hoang et al., 2019) for water and cell culture media respectively. A temperature of 25°C was used for suspensions in water and 37°C for suspensions in media. The samples were left to equilibrate for 120 s at temperature, prior to size measurement.

Immediately before the experiments, samples of monomeric soluble ASC^PYD were centrifuged at 20,000 g at 4 °C for 30 min and filtered with 0.1 μM filter (Millipore). The protein concentration was adjusted to 25 μM by dilution from a higher-concentrated stock solution. Filament formation was triggered by rapid dilution to neutral pH. Thereby, 70 μl of monomeric ASC^PYD was mixed with 0.45 μl of 2.75 M NaOH solution to a reach the pH of 7.5. The solution was mixed at room temperature by careful pipetting, to avoid introduction of air bubbles, and immediately transferred to a quartz cuvette with 1 cm path length. Between runs, cuvettes were carefully cleaned with 1 M Hellmanex solution (Sigma-Aldrich) to avoid cross-seeding effects between sequential measurements. Filament growth was monitored by dynamic light scattering with a Malvern Zetasizer Nano ZS series instrument. The laser focal spot was positioned in the middle of the cuvette and maintained fixed for all the measurements. To maximize the intensity of the scattered light, the minimal attenuation level was used. Data were acquired in 60 s intervals by averaging three runs of 20 s, until a total time of 350 min. Afterwards, the protein solution was blotted on EM grids, negatively stained and imaged with transmission electron microscopy to visualize filament formation.