S3000 sonicator

The harvested cell pellets were resuspended in lysis buffer (50 mM Tris-acetate, 300 mM NaCl, pH 7.4). Then, cells were disrupted on an ice bath by sonication using Misonix S3000 sonicator with the following program: total ON time 1-5 min; 5s ON/10s OFF; Power 12 W. Following, the cell lysate was centrifuged at 18,000×g and 4 °C for 20 min to remove cell debris.

The preparation of PrP seeds and substrates, as well as PMCA, were conducted as previously described [11 (link), 40 (link)] and above. Each seed was diluted in the substrate at a ratio of 1:100 (1 µL seeds + 99 µL substrates containing 50 µg/mL heparin) into a 200 µL PCR tube with 1 PTFE beads (diameter 3/32’’) (Teflon, APT, RI). 20 µL of each mixture was taken and kept at − 20 °C as a non-PMCA control. The remaining mixture was subjected to serial PMCA (sPMCA). Each cycle was comprised of a 20 s elapse time of sonication at amplitude 85 (250 watts; Misonix S3000 sonicator) followed by an incubation period of 29 min 40 s at 37 °C. Each round of sPMCA consisted of 96 cycles. For subsequent sPMCA rounds, 10 µL sample was taken from the last cycle of the immediate preceding round and placed into 90 µL fresh normal brain substrates to start a new round of amplifications.

Propidium iodide (PI, MW = 668.4) was added to the Krebs solution in the presence or absence of the test agent/agents at the beginning of stretch application and at a concentration of 25 µM. After cells were subjected to a specified duration of stretch stimulus, they were further incubated to complete a total of 30 min uptake time including the stretch duration. The cells were then washed 3 times with control Krebs solution and homogenized for 1 min (4 strokes of 15 s at 5 s intervals) using Misonix S3000 sonicator at a 6 W power setting (Misonix, New York, NY, USA) in 250 μL of distilled water. Protein in the sample was determined using a bicinchoninic acid assay [37 (link)] and PI was measured in 100 µL of homogenate transferred to a flat clear bottomed black plate using a SpectraMax iD5 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA) by quantifying fluorescence at excitation and emission wavelengths of 535 nm and 617 nm, respectively. The control level of PI fluorescence was somewhat variable in experiments carried out using a different batch of cells on a different day. For this reason, we expressed PI results relative to the specific control for each set of experiment. The results are expressed as relative fluorescence/mg protein.