Borate buffer

For this in tubo assay, in 96-well microplates, 90 μL of a solution of soybean lipoxygenase from Glycine max type I-B (Merck) at 560 U/mL in a borate buffer (0.2 M pH = 9, Thermo Fisher Scientific) were distributed in each well (enzyme’s final concentration per well: 252 U/mL). Fifty microliters of the samples (final concentration per well: 500 μg/mL) were added and completed with 50 μL of the borate buffer. Fifty microliters of (-)-Epigallocatechin gallate, 95%, (Acros Organics) were used as a positive control (final concentration per well 250 μg/mL). The positive control and samples were prepared in ultrapure water and filtered on a 0.20 μm cellulose acetate membrane. The plate was sealed and incubated at 25 °C for 10 min. Ten microliters of linoleic acid (1 mM emulsified in ultrapure water: ethanol, 9:1, v/v, Merck) were then added to each well. A first absorbance reading was performed with a microplate reader at 234 nm, and successive readings were performed for 10 min at 25 °C to assess the percentage of inhibition.
Inhibition activity was calculated from the slope obtained from the regression line by the following formula: $\mathrm{I} (\%)=(\frac{\mathrm{Ablank} – \mathrm{Asample} }{\mathrm{Ablank}})\times 100$
where A_blank is the slope obtained for the enzyme in the presence of ultrapure water, and A_sample is the slope obtained for the enzyme in the presence of inhibitor/sample. Triplicate measurements were performed.

The maintenance and differentiation of i³ neurons were conducted following published protocols^{39 (link)}. Wild-type iPSC cells were seeded in plates coated with Matrigel (Corning, 354277) containing induction medium supplemented with 2.5 µM of the Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Tocris Bioscience, 1254). The medium was replaced with fresh induction medium containing doxycycline, but without Y-27632, for two consecutive days. The desired number of Day 3 neurons were seeded in cortical culture medium^{39 (link)} onto plates coated with poly-l-ornithine (Sigma, P3655) diluted in borate buffer (Thermo Fisher, 28341). Half of the medium was replaced every 3–5 days (96-well plate; 1 × 10⁴ cells per well). The isolation and culture of mouse primary cortical neurons was described previously^{7 (link)}. Lentivirus was added to the i³ neurons 15 days after induction and to mouse primary neurons 5 days after in vitro culture. The medium was aspirated and replaced with fresh culture medium the day after infection. Experiments were performed 24–72 h after infection.

100 nm carboxylate-modified FluoSpheres^TM (red fluorescent, Ex/Em 580/605 nm), 10 µm FluoSpheres (green fluorescent, Ex/Em 468/508 nm) and 20x Borate Buffer were purchased from ThermoFisher Scientific (Waltham, MA USA). Endotoxin-free water was obtained from G-Biosciences (St. Louis, MO, USA). Poly(ethylene glycol)-amine (mPEG-Amine, 2 kDa MW) was purchased from Creative PEGWorks (Chapel Hill, NC USA). Other chemicals or reagents were purchased from Sigma-Aldrich (St. Louis, MO USA) unless specified.