N lauroylsarcosine sarkosyl

To determine the prion infectivity, an endpoint titration assay was conducted. The mouse brain homogenate was serially diluted with PBS, and then each dilution was intracerebrally inoculated into 5 or 6 ICR mice. Infectious units (LD₅₀ per gram of the brain) were determined according to Behrens-Karber's formula.³⁵ (link) In some experiments, the brain homogenate was solubilized with 1% N-lauroylsarcosine (Sarkosyl, Sigma-Aldrich, Japan) and 1% n-dodecyl-β-d-maltoside (Dojindo Laboratories, Kumamoto, Japan) for 15 min at room temperature (R/T) and then digested with PK (Roche Diagnosis Japan, Tokyo, Japan) under the following conditions: 5 or 40 µg/ml at 37°C for 30 min, or 100 µg/ml at 37°C overnight. The samples were subjected to western blot for the detection of PrP^Sc as described below. The aliquots of these samples were also diluted to 1:100 in PBS and then subjected to the incubation time assay³⁶ (link) to compare the infectivity.

Stock preparations of amyloid fibrils [4 to 5 mg/ml in tris-buffered saline (TBS)] were diluted to a final concentration of 0.1 mg/ml in TBS. The equivalent of the quantity of recombinant human α-syn fibrils used for the treatment of one petri dish of primary neurons (1.66 μg) was diluted in 500 μl of final volume with solubilization buffer (SB): 10 mM tris (pH 7.5), 150 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, cOmplete EDTA-free protease inhibitors (Roche), and PhosSTOP phosphatase inhibitors (Roche), with a final concentration of 1% (w/v) N-lauroyl-sarcosine (sarkosyl, Sigma-Aldrich), 2 mM MgCl₂, and Benzonase nuclease (~0.5 U/μl; Millipore). For treated primary neuron solubilization, 500 μl of SB was added per petri dish at room temperature. Cells were scraped with a scraper before transferring the lysate in a 1.5-ml centrifuge tube. Fibrils or primary neuron lysates were then solubilized by incubating at 37°C under constant shaking at 600 rpm (ThermoMixer, Eppendorf) for 45 min.