Clear 96 well plate

The NR activity was measured as the rate of nitrite production determined with a spectrophotometric assay (Frungillo et al., 2014 ). All samples were protected from light. Total protein from seedlings was extracted in 50 mM HEPES-KOH pH 7.5, 0.5 mM EDTA, 100 µM FAD, 5 mM Na₂MoO₄, 6 mM MgCl₂, protease inhibitor cocktail (Roche), and the homogenate was incubated for 10–15 min on ice with periodic vortexing. After centrifugation, the supernatant was rebuffered using 10K Amicon Ultra 0.5 centrifugal filter units (Millipore, No. UFC501008) and the protein concentration was measured with Bradford reagent. The protein activity assay was performed in a clear 96-well plate (Greiner) in a total reaction volume of 200 µl. The reaction mixture consisted of 1 mM KNO₃, 1 mM NADH, and either 2 mM EDTA or 6 mM MgCl₂, and was incubated for 55 min at room temperature with gentle shaking. Then, a 1:1 mixture of 1% (w/v) sulfanilamide in 1.5 M HCl and 0.02% (w/v) N-(1-naphthyl)-ethylenediamine dihydrochloride in 1.5 M HCl was added. After 15 min incubation at room temperature, the A₅₄₀ was measured with the TECAN Reader Infinite M1000pro spectrophotometer. The activity in the presence of EDTA represents the total NR activity and the activity, measured in the presence of MgCl₂ represents the actual NR activity.

THP-1 cells were plated out at a density of 3·10⁵ cells/mL in a clear 96-well plate (Greiner) that was either supplemented with sapphire discs (STEM tomography) or coverslips (light microscopy). The cells were differentiated in RPMI 1640 medium (GE healthcare) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco), 1% (v/v) Antibiotic-Antimycotic solution (Gibco) and 50 ng/mL phorbol 12-myristate 13-acetate (PMA, Sigma) at 37 °C and 5% CO₂. After 48 h cells were incubated in PMA-free medium for 6–8 h before the medium was replaced again with PMA-free medium containing 100 or 200 μg/mL Aβ(1-40) and further incubation for 48 h. In a next step, cells grown on a coverslip were stained with CR and analyzed by light microscopy or cells grown on sapphire discs were embedded in epoxy resin and prepared for STEM tomography.

The Cytotoxicity Detection Kit^Plus (LDH, Roche Diagnostics) was used to evaluate the cytotoxicity induced by the PBNs. After the PBN incubation (36 h), at least one well was used as a LDH positive control (100% toxicity) by adding Triton X-100 (Sigma-Aldrich) to a final concentration of 0.1% (~ 15 min incubation at 37 °C). Afterwards, 50 µL supernatant of each well were transferred in a new clear 96-well plate (Greiner) and completed with 50 µL/well of the “dye solution/catalyst” mixture as recommended by the manufacturer. The plate was then incubated in the darkness for 30 min at room temperature. Reaction was stopped by adding 25 µL/well of HCl (1 N) before measuring the absorption at 490 nm using the POLARstar Omega plate reader (BMG Labtech). Relative toxicity (%) was calculated with the following formula: [(exp. value–value non-treated cells)/(value triton–value non-treated cells)] × 100.