Tissue master 125

Ears from 3 days p.i. were snap frozen in 1.5 mL microcentrifuge tubes in liquid nitrogen and stored at -80°C. Frozen ears were transferred to 5 mL round bottom polystyrene tubes in 300 μL of Cell/Tissue Extraction Buffer [100 mM Tris, pH 7.4 (RPI Corp.), 150 mM NaCl (RPI Corp.), 1 mM EGTA (Fisher Scientific), 1 mM EDTA (Fisher Scientific), 1% Triton X-100 (Sigma), 0.5% sodium deoxycholate (Sigma-Aldrich)] with 1 mM protease inhibitor cocktail (Roche). Ears were homogenized using a Tissue Master 125 (OMNI International) and agitated on a rotating platform for 2 hours at 4°C. Tissue homogenates were microcentrifuged for 20 minutes at 13,000 rpm at 4°C and the supernatants were transferred into clean 1.5 mL microcentrifuge tubes and stored at -80°C. A murine IL-1β ELISA (R&D Systems) was performed on collected supernatants from homogenized ears and read using the FLUOstar Omega plate reader (BMG Labtech).

Samples of whole Artemia and rotifers, and samples of larvae head (brain tissue and eyes) were tested for the presence of Betanodavirus. Pooled samples were homogenized with Earle’s balanced salt solution (Hyclone Laboratories Inc., Logan, Utah, USA) supplemented with antibiotics (amphotericin B 200 µg ml⁻¹, gentamycin 500 µg ml⁻¹, penicillin 1000 units ml⁻¹ and streptomycin 1000 units ml⁻¹) using a Tissue Master 125 (OMNI International, Kennesaw, Georgia, USA). After centrifugation of the homogenates at 2000g for 20 min at 4 °C, the supernatants were transferred to new tubes and incubated for 6 h at room temperature. Next, supernatants were inoculated (diluted at 10⁻¹ and 10⁻²) in duplicate onto 48-well plates of semiconfluent E-11 monolayers. Infected cells were incubated at 25 °C and examined daily for the presence of CPE. After 10 days, positive and negative samples were subcultivated (by inoculating 100 µl of the scraped cell suspension onto new cultures) for 15 days.

Muscle samples (~200 mg) were homogenized in 1 mL of phosphate buffered saline (pH 7.4) to extract proteins (the most soluble are cytosolic proteins), using a Tissue Master 125 homogenizer (Omni International, Kennesaw, USA) on ice-cold conditions. The crude homogenates were then centrifuged for 15 min at 10,000 × g and the supernatants were frozen immediately (−80°C) until further analyses.

A volume of 1 mL of titrated crude virus (SGWak97 strain) was mixed with 1 g of non-infected gilthead seabream brain (the fish were obtained from a fish market and checked by RT-qPCR before use). The mixture was homogenized using a Tissue Master 125 homogenizer (OMNI International, Kennesaw, USA) and incubated at 25 °C for 24 h. Afterwards, the tissue was pelleted at 3000× g for 20 min and both phases, supernatant and pelleted tissue, were titrated separately as described above. In parallel, non-infected seabream brain was similarly processed (suspended in one volume of L-15 medium) to prepare the serial w/w dilutions of the infected pelleted tissue, starting with a mixture of 400 µL of infected pellet + 400 µL of non-infected tissue, and continuing with dilutions of 1/5, 1/10, 1/100, and 1/50 using non-infected tissues). From each dilution, 200 µL were subjected to RNA extraction and RT-qPCR amplification as described above.

All animal procedures were IACUC approved and performed in the Biological Resources Laboratory at the University of Illinois at Chicago. Overnight bacterial overnight cultures were diluted 1:20 into fresh media and grown to an OD₆₀₀ ∼0.6. 1 ml of culture (corresponding to 6x10⁸ CFU/ml) was washed, diluted and resuspended in PBS to a final concentration of 1x10⁵ CFU/ml. 8–10 week old female Swiss Webster mice (Charles River Laboratories, Chicago, IL) were injected with 200 ul PBS containing 2x10⁴ CFU L. monocytogenes via the tail vein. At 24, 48 and 72 hrs post-infection, mice were sacrificed and livers and spleens were harvested. Organs were homogenized with a Tissue Master 125 homogenizer (Omni International, Kennesaw, GA) and dilutions were plated onto BHI streptomycin (200 ug/ml) plates. Non-paired student t-test was used for statistical analysis.

Germinated seedlings from each replicate per treatment were collected and then incubated in hot isopropanol for 15 min, following homogenization with an OMNI tissue homogenizer (Tissue Master 125, OMNI International, Kennesaw, GA, USA). The homogenate was used for further lipid extraction and analysis by adapting and updated version of the Bligh and Dyer method was used for lipid extraction [57 (link)]. A total of 10 mg of the sample was transferred to glass centrifuge tubes, and the following solvents were added: 1 mL of methanol containing 0.01% butylated hydroxytoluene (BHT), 1 mL chloroform and 0.8 mL water. The sample with the solutions was homogenized again using an OMNI tissue homogenizer. The sample mixture was centrifuged at 5000 rpm for 15 min. After centrifugation, the organic layer (bottom of the vial) containing the lipids were transferred to pre-weighed 4 mL sample vials with PTFE lined caps (VWR, Mississauga, ON, Canada). The organic layer was dried under a nitrogen stream, and then weighed to determine the amount of lipids recovered [57 (link)]. The recovered lipids in each vial were re-suspended in 1 mL chloroform:methanol (1:1 v/v) and stored at −20 °C for further analysis.