Fluostar optima reader

Rhodamine measurement was used to assess mitochondrial membrane potential integrity. Therefore, fluorescence quenching of rhodamine 123 (Thermo Fisher Scientific, Waltham, MA, USA) was measured over 40 min, as described previously [24 ]. Mitochondria were plated in a black 96-well plate with 75 µg mitochondrial protein per well and diluted in a buffer containing 200 mM sucrose, 10 mM MOPS-Tris, 5 mM succinate, 1 mM Pi(H₃PO₄), 10 µM EGTA, and 2 µM rotenone (pH 7.3). Loss of mitochondrial membrane potential was induced by FCCP (500 nM), which was injected at the end of the measurement. Fluorescence (excitation: 485 nm/12, emission: 530 nm/10) was measured with a FLUOstar OPTIMA reader (BMG Labtech, Ortenberg, Germany).

ATP levels in human dopaminergic neurons upon aSYN overexpression were analysed by luminescence detection according to the manufacturer’s protocol from ViaLight TM plus kit (Lonza, Verviers, Belgium). LUHMES cells were cultured and differentiated on white 96-well plates (10,000 cells/well, Greiner Bio-one, Frickenhausen, Germany). After 72 h of aSYN expression, ATP levels were analysed by luminescence detection using a FLUOstar OPTIMA reader (BMG Labtech, Offenburg, Germany).

Real Time Quaking-Induced Conversion (RT-QuIC) was used to detect the presence of PrP^Sc in cerebrospinal fluid (CSF) of patients, as previously described [11 (link)]. Briefly, 15 μl of each CSF was added to 85 μl of reaction mix in black, 96-well microplates. Samples were tested in quadruplicate together with positive (definite CJD) and negative (non-CJD) controls. The RT-QuIC reaction mix was prepared as follow: 300 mM NaCl, 10 mM phosphate buffer at pH 7.4, 1 mM ethylenediaminetetraacetic acid tetrasodium salt dehydrate (EDTA) at pH 8.0, 0.002% of Sodium dodecyl sulfate (SDS), 10 μM thioflavin-T (ThT) and 0.1 mg/ml of Syrian hamster recombinant truncated form of prion protein (Ha rPrP 90–231). After sealing, the plate was incubated in a FLUOstar OPTIMA reader (BMG Labtech) at 55 °C, over a period of 60 h with intermittent cycles of shaking (1 min) double-orbital (600 rpm) and incubation (1 min). The fluorescence intensity, expressed as arbitrary unit (AU), was taken every 15 min using 450 ± 10 nm (excitation) and 480 ± 10 nm (emission) wave-lengths. A sample was considered positive if the mean of the highest two fluorescence values (AU) of the replicates was higher than 10.000 AU and at least two out of four replicates crossed the threshold set at 60 h. The sample was considered negative if none or only one replicate (out of four) crossed the threshold before 60 h.

The determination of ATP levels was performed using the ViaLight Plus Kit (Lonza, Verviers, Belgium) according to the manufacturer’s protocol. Briefly, cells were lysed, transferred to a white-walled 96-well plate and the luciferase containing ATP monitoring reagent was added. The resulting luminescence was detected with a FLUOstar OPTIMA reader (BMG Labtech, Ortenberg, Germany).

Cell viability was quantified by the reduction of MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) by the LUHMES cells for 1 h in the cell culture incubator. Total medium containing MTT was removed, and the cell culture dishes were frozen at −80 °C for at least an hour to stop the reaction. After thawing at room temperature, the dye was dissolved in 80 µl of DMSO, and the absorbance was measured at 570 nm with a reference filter at 630 nm with a FLUOStar OPTIMA reader (BMG Labtech, Ortenberg, Germany) as described^{29 (link)}.
Additionally, to analyse the cell viability at different time intervals, we also measured the lactate dehydrogenase (LDH) release in the media after 24, 48, and 72 h of viral infections by using commercially available LDH-Glo^TM Cytotoxicity assay (J2380).

The oxidation of protein results in the production of stable carbonyl groups, which are used as a measure of oxidative injury. Carbonyl content was determined with the Protein Carbonyl Content Assay Kit (Sigma MAK094, Merck KGaA, Darmstadt, Germany) according to the manufacturer’s protocol. In brief, hypothalamic tissue samples (20.0 ± 7.9 mg) were dissolved in dH2O, homogenized with a T10 basic Ultra-Turrax (IKA-Werke, Staufen, Germany), and sonicated for 10 min in a water bath at rt. Afterwards, the homogenates were centrifuged for 15 min at 13,000× g and 4 °C resulting in the protein-comprising supernatants (Heraeus Fresco 17; Thermo Fisher Scientific, Darmstadt, Germany). Next, the samples were treated with 1% streptozocin followed by successive incubation with 2,4-dinitrophenylhydrazine (DNPH), TCA, acetone, and guanidine. Absorbance was measured at 355 nm (FLUOstar OPTIMA reader, BMG Labtech, Ortenberg, Germany). To determine the amount of protein per sample, a Pierce BCA Protein Assay (Thermo Fisher Scientific, Darmstadt, Germany) was performed with 2 µL of the remaining solution.
The carbonyl content was calculated as follows: $$\mathrm{C}\mathrm{P}\left(\mathrm{n}\mathrm{m}\mathrm{o}\mathrm{l} \mathrm{c}\mathrm{a}\mathrm{r}\mathrm{b}\mathrm{o}\mathrm{n}\mathrm{y}\mathrm{l}/\mathrm{m}\mathrm{g} \mathrm{p}\mathrm{r}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{i}\mathrm{n}\right)=\frac{(\frac{{\mathrm{A}}_{355}}{6.364}\times 95)}{\mathrm{P}}\times 1000$$
where 6.364 = extinction coefficient; 95 = total volume in well (µL); P = amount of protein in 95 µL sample; 1000 = conversion factor (µg to mg).