Gen5 software version 2

Cell lines and cell culture: cAMP Hunter^TM Chinese hamster ovary cells (CHO-K1) that express human μ-opioid receptor (OPRM1), human κ-opioid receptor (OPRMK1), and human δ-receptor (OPRMD1) were purchased from Eurofins DiscoverX (Fremont, CA, USA). Cell culture was performed as previously described [18 (link)]. Briefly, cells were plated in a 384-well white tissue culture microplate at 10,000 cells/well density and incubated overnight at 37 °C in 5% CO₂. Compounds were first dissolved in DMSO to form stock solutions (5 mM concentration), and then 9 doses of 100× solutions were prepared by serial dilution with DMSO. Then, 5× solutions were prepared by diluting 100× solutions with buffer consisting of Hank’s Buffered Salt Solution, HEPES, and forskolin. In the agonist assay, cells were treated with compounds (at 1× final concentration) and incubated at 37 °C for 30 min. In the antagonist assay [17 (link)], cells were pretreated with compounds for 15 min at 37 °C followed by 30 min incubation at 37 °C with selected agonists at their EC₅₀ or EC₉₀ dose. The HitHunter cAMP Assay for Small Molecules by Eurofins DiscoverX (Fremont, CA, USA) was then used according to manufacturer’s directions and the BioTek Synergy H1 hybrid plate reader (BioTek, Winooski, VT, USA) and Gen5 Software version 2.01 (BioTek, Winooski, VT, USA) were used to quantify luminescence [19 (link)].

Assays were performed as previously described [26 (link)]. Briefly, the cAMP Hunter cells were plated in a 384-well white tissue culture microplate at a 10,000 cells/well density and incubated overnight at 37 °C. Compounds were first dissolved in DMSO to form 5 mM stock solutions, and then 9–10 doses of 100X solutions were prepared by serial dilution with DMSO. Subsequently, these 100X solutions were further diluted with assay buffer consisting of Hanks’s buffered salt solution, HEPES, and forskolin to generate the 5× working solutions. In the agonist assay, cells were treated with compounds (at 1× final concentration) and incubated at 37 °C for 30 min. In the antagonist assay [22 (link)], cells were pretreated with compounds for 15 min at 37 °C followed by 30 min incubation at 37 °C with selected agonists at their EC50 or EC90 dose. The HitHunter cAMP Assay for Small Molecules by Eurofins DiscoverX (Fremont, CA, USA) was then used according to the manufacturer’s directions and the BioTek Synergy H1 hybrid and Cytation 5 plate readers (BioTek, Winooski, VT, USA) and Gen5 Software version 2.01 (BioTek, Winooski, VT, USA) were used to quantify luminescence.

Absorbance measurements in turbidity assays were performed in 96-well microplates at 450, 500, 550, 600, and 650 nm, at 25 °C or 37 °C according to the experimental assay, with a microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA) managed by the Gen 5 software version 2.01 provided by BioTek Instruments, Inc. The plates were linearly agitated for 30 s before the measurement.
Samples were prepared to a final volume of 100 μL in each well. All assays were performed in triplicate. The absorption values of the negative controls were determined and subtracted from the corresponding raw data. The final percentage of aggregation was calculated as an average of the wavelengths measured and represented with the corresponding standard deviation. The extent of amyloid fibril formation was assigned as 100% at the end of the TTRwt aggregation experiment.

Briefly, cells were plated at 10,000 cells/well density in a 384-well tissue culture plate and incubated overnight at 37 °C in 5% CO₂. Stock solutions of compound were made in 100% DMSO at a 5 mM concentration. A serial dilution of 10 concentrations was made using 100% DMSO, creating 100X working solutions of the compound for treatment. The 100X working solutions were then diluted to 5X working solutions using assay buffer consisting of Hank’s Buffered Salt Solution, HEPES, and forskolin. For the agonist assay, cells were incubated at 37 °C with compounds for 30 min at a 1X final concentration. For the antagonist assay, cells were incubated at 37 °C with compounds for 15 min before 30-min incubation at 37 °C with selected agonist at their EC₅₀ or EC₉₀ dose. Detection was made by using the HitHunter cAMP Assay for Small Molecules by DiscoverX according to manufacturer’s directions, and the BioTek Synergy H1 hybrid plate reader (BioTek, Winooski, VT, USA) and Gen5 Software version 2.01 were used to quantify luminescence (BioTek, Winooski, VT, USA) [16 (link)].

Cell lines and cell culture: cAMP Hunter^TM Chinese hamster ovary cells (CHO-K1) that express human μ-opioid receptor (OPRM1), human κ-opioid receptor (OPRMK1), and human δ-receptor (OPRMD1) were purchased from Eurofins DiscoverX (Fremont, CA) and used for the forskolin-induced cAMP accumulation assays [28 (link)].
Briefly, cells were plated at 10,000 cells/well density in a 384-well tissue culture plate and incubated overnight at 37 °C in 5% CO₂. Stock solutions of compound were made in 100% DMSO at a 5 mM concentration. A serial dilution of 10 concentrations was made using 100% DMSO, creating 100× solutions of the compound for treatment. The 100× solutions were then diluted to 5× solutions using assay buffer consisting of Hank’s Buffered Salt Solution, HEPES, and forskolin. For the agonist assay, cells were incubated at 37 °C with compounds for 30 min at a 1× concentration. For the antagonist assay, cells were incubated at 37 °C with compounds for 15 min before 30-min incubation at 37 °C with selected agonist at their EC₅₀ or EC₉₀ dose. The HitHunter cAMP Assay for Small Molecules by DiscoverX was then used according to manufacturer’s directions and the BioTek Synergy H1 hybrid plate reader (BioTek, Winooski, VT, USA) and Gen5 Software version 2.01 (were used to quantify luminescence (BioTek, Winooski, VT, USA) [17 (link)].

The peroxidation of serum lipid components was monitored by following the kinetic of conjugated dienes formation at 234 nm after exposure to copper sulfate. The method was optimized in a microplate reader (Synergy HT, Biotek Instruments, Winooski, VT, USA). Briefly, serum was centrifuged at 10,000 g for 2 min and 1:100 in PBS 5 mM sodium citrate 18 mM. Reaction was carried out at 37 °C and was started by the injection of CuSO4 freshly prepared at a final concentration of 25 μm in distilled water. Sigmoidal kinetic was recorded for 6 h every 5 min. The most representative indexes of sample resistance to peroxidative insult were calculated using GEN5 software version 2.0 (Biotek Instruments, Winooski, VT, USA): namely, the length of initiation phase (lag time), rate of dienes production (Vmax), and delta OD (optical density) corresponding to the blank subtracted plateau.