Axygen sealing tape

A fibrillization assay was performed using 100 μL of 1 mg/mL monomeric αSYN (rPeptide). Samples containing either αSYN alone or αSYN + NPs at 1:10 volume ratio were loaded with 20 μM Thioflavin T (ThT) (Acros Organics) into 96 well clear bottomed plates (Corning), sealed with Axygen sealing tape (Corning), and shaken at 600 rpm at 37°C for at least 63 h. Samples were taken at various time points and a POLARstar Omega plate reader (BMG Labtech) was used to monitor the increase in ThT intensity, with fluorescence intensity measured at excitation −450 nm and emission −485 nm. This protocol was adapted from the literature (Moriarty et al., 2017 (link)).

Lyophilized acetylated αS or βS was dissolved in 10 mM PBS (pH 7.4), and large aggregates were removed by centrifuge filtration (50 kDa MWCO, Millipore Sigma, St. Louis, MO). The dissolved protein was concentrated in 3 kDa centrifuge units (Millipore Sigma, St. Louis, MO) to 1 mg/mL (αS) or 3 mg/mL (βS). To create fibrils, 100 uL of each sample mixture was loaded into 96-well clear bottom plates (Corning, Corning, NY) with a single Teflon bead (3 mm, Saint-Gobain N.A., Malvern PA). The plates were sealed with Axygen sealing tape (Corning, Corning, NY) and shaken at 600 rpm and 37 °C in a POLARstar Omega fluorimeter (BMG Labtech, Cary, NC). Fibrils were allowed to form for at least 72 hours. Samples used for AFM, ESI-MS, ssNMR, and cell toxicity and shedding experiments were collected by centrifugation at 14k rpm for 2 hours, and washed through multiple rounds of re-suspension in 10 mM PBS (pH 7.4) and centrifugation at 14k rpm for 2 hours in order to remove residual soluble and non-fibrillar components.

The effect of AM-NPs on Aβ fibrilization was assessed as previously described [59 (link)]. Samples containing 8 µM of monomeric Aβ in the absence or presence of NPs (1:10, v/v) were loaded with 20 µM Th-T into 96-well clear bottomed non-binding half-area plates (Corning). NPs only mixed with Th-T were included as a negative control to measure any background reading of NPs. Plates were sealed with the Axygen sealing tape (Corning) and the fluorescence intensity was monitored over 63 h using a Tecan Infinite M200 Pro microplate reader with excitation at 450 nm and emission at 485 nm while agitated at 600 rpm at 37 °C. After subtraction of NP background fluorescence, each sample containing Aβ and NPs was normalized to the Aβ fluorescence.