Fluoroskan ascent microplate reader

The oxygen radical absorbance capacity (ORAC) assay was performed to assess the antioxidant capacity of MEs, using Trolox as a positive control [26 (link)]. Briefly, 25 μL of MEs or Trolox were pipetted in a 96-well white microplate (with clear bottom), whereas phosphate buffer (25 μL, 75 mM, pH 7.4) was transferred in the negative control wells. Fluorescein solution (150 μL, 25 nM) was added in each well and the plate was incubated in the dark at 37 °C for 30 min. Subsequently, AAPH solution (25 μL, 0.15 M) was added in the positive control and the antioxidant wells, whereas phosphate buffer (25 μL) was pipetted in the negative control, and the fluorescence was measured every 2 min over a period of 2 h (485/20 nm excitation, 525/20 nm emission) using a Fluoroskan Ascent microplate reader (ThermoScientific, Waltham, MA, USA). The non-enzymatic DHA oxidation that occurred in the microemulsions was measured as described below (Section 2.4.4).

A drug library comprising 1,165 off-patent drugs was used for identifying inhibitors of the candidate compounds with higher effectiveness than MMC. The mTCFs were placed onto 96-well plates (3,000 cells/mm2) with a Multidrop Combi (Thermo Fisher Scientific, USA) and incubated at 37 °C overnight. They were then treated with DMEM containing the candidate drugs at a final concentration of 10 μM. Twenty-four hours later, the cells were incubated in DMEM containing 10% Alamar Blue reagent (Life Technologies Inc., USA) at 37 °C for 2 h in the dark. Fluorescence intensity was measured at 544 nm excitation and 590 nm emission with a Fluoroskan Ascent microplate reader (Thermo Fisher Scientific, USA).
For the evaluation of corneal cytotoxicity, the human corneal epithelial cells were placed onto 96-well plates at 2,000 cells/mm2 and treated with 10 μM mTCF-growth suppressive drugs in CnT-20 medium at 37 °C for 24 h. All following procedures were then performed as above.

The solution containing in vitro generated α-synuclein aggregates was sonicated for 3 min at 500 W and serially diluted (from 10^− 1 to 10^− 12 volume/volume) in its own reaction buffer. Five μL of the following dilutions: undiluted, 10^− 3, 10^− 6, 10^− 9, 10^− 12 was added to 95 μL of reaction mix and subjected to RT-QuIC analysis. Reaction mix was composed as follow: rec-αS diluted in 40 mM PBS (pH 8.0), 170 mM NaCl and 10 μM Thioflavin-T (ThT) at the final concentration of 140 μM. All reagents used for the preparation of the reaction mix were filtered through a 0.22 μm filter before the addition of α-synuclein aggregates. All RT-QuIC reactions were performed in triplicate in a black 96-well optical flat bottom plate (Thermo Scientific) using the Fluoroskan Ascent microplate reader (Thermo Scientific). Samples underwent to cycles of shaking (1 min at 600 rpm, single orbital) and incubation (14 min at 42 °C). Fluorescent intensities, expressed as arbitrary units (AU), were taken every 30 min using 450 ± 10 nm (excitation) and 480 ± 10 nm (emission) wave-lengths, with a bottom read. The addition of a 3-mm glass bead (Sigma) was necessary to sustain protein aggregation.

Rec-αS was diluted in a reaction mix composed of 40 mM PBS (pH 8.0), 170 mM NaCl and 10 μM Thioflavin-T (ThT) at the final concentration of 140 μM. Reactions were performed in triplicate in a black 96-well optical flat bottom plate (Thermo Scientific). Each well was supplemented with 100 μL of reaction mix. The plate was sealed with a sealing film (Thermo Scientific), inserted into a Fluoroskan Ascent microplate reader (Thermo Scientific) and subjected to cycles of shaking (1 min at 600 rpm, single orbital) and incubation (14 min at 42 °C). The addition of a 3-mm glass bead (Sigma) was required to sustain protein aggregation. The presence of protein aggregates was confirmed by means of ThT analysis, Western blot and Transmission Electron Microscopy (TEM) analyses.