Centrifuge 5415d

The detection of PK-resistant PrP from inoculated mouse brains was performed following two different protocols: i) a standard procedure as described above and ii) an adapted protocol developed to detect atypical PrP^Sc. Briefly, 40 μl of 10% brain homogenate were diluted in 160 μl phosphate buffered saline (PBS, Fisher Bioreagents) and digested with 12.5 μg of PK in digestion buffer (8% Sarkosyl and 5% sodium dodecylsulfate in PBS). The sample was incubated for 15 min at 37 °C with constant agitation at 500 rpm (Thermomixer comfort, Eppendorf) and then sonicated for 8 × 30 seconds (Misonix Sonicator Q-700 microplate system, Qsonica). The sample was mixed with 200 μl of butanol (Sigma-Aldrich) and centrifuged for 7 min at a 15,000 g (5415D centrifuge, Eppendorf). The supernatant was removed and the pellet resuspended and incubated 5 min at room temperature with 50 μl of Laemmli loading buffer (Bio-Rad). Before loading, the samples were incubated for 5 min at 100 °C, centrifuged for 15 min at 15,000 g and the supernatants collected for further analysis by Western blotting as described above using 12B2 antibody [diluted 1:2,500 in 0.1% (w/v) of fat-free powder milk in PBS (Fisher Bioreagents) with 0.05% Tween-20 (Sigma-Aldrich)].

The pathogenic strain VPAP30 was recovered from a massive mortality event of reared-larvae of the scallop A. purpuratus occurring in a commercial hatchery located in Tongoy Bay in the north of Chile. Triplicate samples of settled dead and moribund larvae were aseptically collected from the bottom of the rearing tank during its water exchange using a sterile glass flask and were transported to the laboratory for immediate processing. Larval samples were centrifuged at 960 g for 2 min using an Eppendorf 5415D centrifuge (Hamburg, Germany) and the water excess was discarded. Settled larvae were ground by hand using a sterile glass digester containing 2 mL of sterile physiological saline (0.85% NaCl; PS) to obtain a homogenate according to the method of Nicolas et al. (1996) (link). The homogenate was inoculated in triplicate onto Tryptic Soy Agar (Difco, NJ, United States) with 2% of NaCl (Oxoid, Hants, United Kingdom) (TSA2), and plates were incubated at 20°C for 48 h. The predominant colony, which grew almost as a pure culture on TSA2 plates, was isolated and preserved at -85°C in CryoBank (Mast Diagnostic, Merseyside, United Kingdom) vials prior use.

For each tomato cultivar, five fruits were selected randomly and chopped. Then, 1 g of peel and 1 g of pulp were weighed and 6 mL (for peel) and 3 mL (for pulp) of 70% acetone were immediately added. Samples were homogenized by Turrax (Ultra-Turrax^® T25 based IKA, Saint Louis, MO, USA) for 5 min, then placed in a sonicator (Elma Transsonic T 460/H, Wezikon, Switzerland) for 15 min and then homogenized again by Turrax. Samples were then centrifuged at 4000× g r.p.m. for 5 min (Eppendorf^® 5415D centrifuge, Hamburg, Germany). Finally, supernatants (i.e., the extract) were transferred to 2 mL Eppendorf tubes.

Bacterial cells from donor and transconjugant selective media plates were suspended in PBS and centrifuged at 10,000× g for 1 min at room temperature in an Eppendorf™ 5415D centrifuge with an F-45-24-11 rotor (Eppendorf™, Hamburg, Germany). The pellet was resuspended in 100 μL of PBS. We used the agarose pad method to obtain images of fluorescent bacteria (donor and transconjugants) [38 (link)]. Briefly, a 1% agarose solution was poured on a plain surface bordered with microscope slides to achieve agar pads of even thickness. After the agarose solidified, coverslip-sized pads were cut using a sterilized scalpel and placed on another microscope slide. A volume of 2–5 μL of bacteria suspended in PBS was spread over the agar pad and covered with a coverslip. A Nikon Eclipse Ni-U upright microscope (Nikon, Tokyo, Japan) was used with bright-field, GFP, and RFP filters to record and analyze the fluorescent bacteria at magnifications of 20× and 40×. For rapid screening of fluorescent bacterial colonies, we took a small number of bacteria directly from the individual colonies grown on the plate using a sterile toothpick and mixed them with 10 μL of PBS or deionized water on a microscope slide. After placing a coverslip and air drying the slide for ten minutes, we observed fluorescent mScarlet and GFP expression in the donors, recipients, and transconjugants under the microscope.

Monoinfected rabbit sera reactive to each serotype of dengue virus (DENV1, DENV2, DENV3 and DENV4), as well as uninfected sera controls, were kindly provided by Dr. Erna G. Kroon from Laboratório de Vírus, ICB/UFMG. To this end, healthy New Zealand male rabbits, at least two months old, were purchased from the Escola de Veterinária of UFMG’s Experimental Farm. For immunization, two milliliters of DENV-infected cells (viral titer between 10⁵ and 10⁶ PFU/mL) were inactivated under ultraviolet light (UV) for 5 min and then diluted in Freund’s Complete Adjuvant (Sigma, St. Louis, MO, USA) at a ratio of 4:4 mL of inoculum. This dilution, after emulsion, was inoculated into each rabbit, at four inoculation points (1 mL/point) subcutaneously. This process was repeated at least twice, at intervals of 22 to 30 days. Boosts were prepared with incomplete Freund’s adjuvant (Sigma, St. Louis, MO, USA). Blood samples were collected between 14 and 21 days after each immunization. The collected blood was centrifuged at 3000 rpm (Eppendorf 5415D Centrifuge, Hamburg, Germany) and frozen at −20 °C until testing.