Thermotop

S. cerevisiae inoculum was prepared in a 96-well plate (Tissue Culture Testplate®, SPL Life Sciences) containing 150 µL defined medium at pH 5. The culture was grown overnight in a thermomixer (Eppendorf ThermoTop®) at 30 °C, 200 rpm. Cells were resuspended by pipetting prior to measurement of the cell density using a plate reader (Spectrostar Nano®, BMG Labtech). The cells were then transferred to a fresh 96-well plate (CR1496e®, System Duetz) with a final volume of 200 µL and starting OD₆₀₀-equivalent of 0.02. Strain analysis was performed in triplicate. The plate was sealed with a machine-specific aerobic sandwich cover (CR1296a EnzyScreen B.V.). The plate was incubated in a growth profiler (GP960 REV2®, EnzyScreen) for 72 h at 30 °C, 230 rpm, and green light scattering measurements were taken hourly. Defined medium was used as a blank, and the value of the green light scattering signal (green value, GV) obtained from this was subtracted from the values measured during cell growth.
Green values were converted into OD₆₀₀ values using a standard curve obtained previously using S. cerevisiae strain CENPK_113.7D^{71 (link)} grown in defined medium. The following two equations were used to convert GV into OD₆₀₀ values:
For GV ≤ 10: $$OD600=0.0322x2.{72}^{\left(0.4328xGV\right)}$$
For GV ≥ 10: $$OD600=-0.0004xG{V}^3+0.0398xG{V}^2-0.6506xGV+5.4063$$

Phase I metabolism reactions were performed in vitro with the use of the HLM fraction. The incubation system consisted of 50 μM substrate, 55 mM phosphate buffer (pH 7.4), and 0.5 mg mL⁻¹ microsomes. The incubation system was pre-incubated at 37 °C for 2 min (Eppendorf ThermoMixer C equipped with Eppendorf ThermoTop, Hamburg, Germany) and then the metabolic reactions were initiated by the addition of 10 μL NADPH (20 mM). Total volume of the reaction suspension was 200 μL and no organic solvent was added into the system. The reaction was terminated after 0, 30, 60, 120, and 180 min of incubation with 200 μL of ice-cold acetonitrile:methanol mixture (1:1). Next, the precipitated samples were centrifuged at 15,000 rpm for 10 min at 4 °C (Eppendorf 5424R, Hamburg, Germany) and the supernatants (50 μL) were transferred into autosampler vials for LC-MS analysis. The negative control samples were prepared as described above without the addition of NADPH solution.

For MN-QuIC, we leveraged a standard benchtop shaking incubator (ThermoMixer) to produce QuIC-based prion amplifications as previously reported by Cheng et al.^{22 (link)} and Vendramelli et al.^{23 (link)}, although with slight modifications. Plates were prepared identically to those amplified on the plate reader (see protocol above). Instead of shaking on a plate reader, reactions were performed on a ThermoMixer® C equipped with SmartBlock plate and Thermotop (Eppendorf, Enfield, Connecticut, USA) at 48 °C for 24 h at 600 RPM (60 s shake and 60 s rest). We selected a 24 h run time based on independent RT-QuIC results for RPLNs and palatine tonsils from CWD positive WTD reported in Schwabenlander et al.²⁷ , including those examined herein, showing significant seeding activity within 9 to 24 h (Fig. S3). The resultant products were visualized with the addition of gold nanoparticles (as described below).

A Nanodrop II liquid handling system (BioNex Solutions, San Jose, CA, United States), was used for all pipetting steps. BioNex Nanodrop II accessories can be mounted on two nests, mostly used for microtitration plates. There are also two positions for trays (containing in this case E. coli suspension and decontaminating bleach solution) or PCR tube holders. E. coli was cultivated using a microplate shaker with a controlled heating platform (ThermoMixer C, Eppendorf) and heated lid (ThermoTop, Eppendorf). For screening, sterile transparent 384-well plates (Corning, United States) were used. Optical densities (OD₆₀₀) and fluorescence intensities of the β-galactosidase-catalyzed reaction product (excitation and emission maxima: 365 and 448 nm, respectively) were measured using an Infinite M1000Pro plate reader (Tecan, CH). In case the HTS automation is not available the method could be downscaled and adapted for manual pipetting similarly as described by Spíchal (2011) (link).

Individual FFR coupons were placed within the wells of 24-well plates and inoculated with either (i) 5 µL of the SARS-CoV-2 NZ/Queenstown/01 strain (10⁷ TCID₅₀/mL) or (ii) 5 μL of S. aureus, A. baumannii, or P. aeruginosa from bacteria culture (1 × 10⁶ CFU) or biofilm (one colony representing 3 × 10⁷ to 5 × 10⁸ CFU). Following a 10 min incubation, the 24-well plate with the FFR coupons was placed on a pre-heated Eppendorf ThermoMixer^® C with a SmartBlock™ (Eppendorf, Hamburg, Germany) and covered with the ThermoTop^® (Eppendorf) to guarantee a constant thermal profile over the entire plate, at different temperatures (22 to 80 °C) and times (15 to 120 min). Finally, we also performed a 24 h stand-down experiment where bacterial biofilms were prepared as above but left for 24 h at room temperature prior to starting the experiment. Control experiments to determine the number of bacteria within the biofilm were determined by the selection of one colony resuspended in 1 mL PBS, diluted and plated onto the respective selective media for each strain (five colonies each), incubated overnight, and colonies counted.