# Effects of Pulsed Electric Fields on Yeast with Prions and the Structure of Amyloid Fibrils
## 2 Materials and Methods
### PEF Permeabilization Assay  
A unique square pulse wave PEF generator was developed at the Institute of High Magnetic Fields (Vilnius Gediminas Technical University Vilnius, Lithuania), generating 100 ns-1 ms pulses. Up to 30 kV was used in this research [32]. PEF experiments were performed in 1 M sorbitol. The impedance of the cuvette was in the range of 9.1 \(\pm\) 0.1 k\(\Omega\), the highest expected current was in the range 0.02 to 0.3 A, corresponding to 2 and 26 kV/cm treatments. Temperature rise was measured using a Pt1000 sensor (InnovativeSensor Technology, Wattwil, Switzerland).  
The summary of PEF treatments, including the energy of each protocol, is presented in Table 1.  
Different prion variants of _S. cerevisiae_ PSI+ and _S. cerevisiae_ psi\(-\) prion-free strains were plated on agarized YPD mediums and grown for 48 h at 30 \({}^{\circ}\)C. One sterile colony was transferred to a tube with 5 mL of a YPD medium and grown in a shaker with aeration for 12 h at 30 \({}^{\circ}\)C, 130 rpm. The overnight culture was transferred again to a sterile 5 mL YPD tube and diluted to the final OD 0.2 and grown for 3-4 h until 0.5-0.6 OD was reached.  
\begin{table}
\begin{tabular}{c c c c c c} \hline
**Electric Field** & **Burst** & **Pulsed** & **Energy** & **Energy Density** & **Temperature** \\
**(kV/cm)** & **Duration** & **Current * (A)** & **(J)** & **(kJ/L)** & **Rise (\({}^{\circ}\)C)** \\ \hline
2 & & 0.02 & 0.02 & 0.4 & 0 \(\pm\) 0.1 \\
4 & & 0.04 & 0.09 & 1.8 & 0.3 \(\pm\) 0.2 \\
6 & & 0.07 & 0.20 & 4 & 0.5 \(\pm\) 0.2 \\
8 & & 0.09 & 0.35 & 7 & 0.8 \(\pm\) 0.2 \\
10 & & 0.11 & 0.55 & 11 & 1 \(\pm\) 0.3 \\
12 & & 0.13 & 0.79 & 15.8 & 1.2 \(\pm\) 0.2 \\
14 & 1 ms \(\times\) 5 (1 Hz) & 0.15 & 1.08 & 21.6 & 1.3 \(\pm\) 0.6 \\
16 & & 0.18 & 1.41 & 28.2 & 1.5 \(\pm\) 0.2 \\
18 & & 0.20 & 1.78 & 35.6 & 2.1 \(\pm\) 0.4 \\
20 & & 0.22 & 2.20 & 44 & 2.3 \(\pm\) 0.5 \\
22 & & 0.24 & 2.66 & 53.2 & 2.8 \(\pm\) 0.4 \\
24 & & 0.26 & 3.16 & 63.2 & 3.6 \(\pm\) 0.9 \\
26 & & 0.29 & 3.71 & 74.2 & 4.6 \(\pm\) 1.2 \\ \hline \end{tabular}  
* Estimated based on Ohm’s law and cuvette impedance of 9.1 k\(\Omega\).  
\end{table}
Table 1: The summary of pulsed electric field (PEF) treatments.  
This indicated a transition of the culture to an exponential growth phase. Then, yeast cells were harvested via centrifugation at 9000\(\times\)_g_ for 90 s. After centrifugation, all of the following steps were performed by keeping the cells cold, on ice, to slow down the metabolic reactions and cell division. To remove unnecessary salts and ions adhering to the cell wall that could increase the sample's electrical conductivity, the cells were washed 3 times with 1.5 mL of cold 1 M sorbitol solution. The washed cells were suspended in 1 mL of 1 M sorbitol to the final concentration of 10\({}^{9}\). After cell suspension was transferred to the 1 mm gap electroporation cuvette and subjected to the PEF ranging from 2 kV/cm to 26 kV/cm, 5 pulses with the duration of 1 ms. After PEF treatment, cells were plated on a fresh medium and grown for 48-72 h, wherein colonies were counted and analyzed for the appearing prions, indicated by the switch of the colony's color. In total, two controls were used in this research: the first control was prepared and plated before the PEF experiments, showing the number of the viable cells in the suspension; the second control had cells that were prepared and stored on ice throughout the duration of the experiment, showing a general survivability during the experimental procedure.