Seakem agarose

The RAW 264.7 cells were seeded into six-well plates at a density of 2 × 10⁶ viable cells per well. On the following day, the B. adolescentis re-suspended in DMEM (Lonza) was used to make ten-fold dilutions from MNV-1 lysate. The RAW 264.7 cells were washed with PBS to remove the antibiotics, the mixture of bacteria and MNV-1 was added onto the cells (500 μl per well, two wells per sample). Plates were incubated for 1 h at room temperature and manually rocked every 15 min before aspirating the inoculum and overlaying the cells with 1.5% SeaPlaque agarose (Cambrex, Rockland, ME, USA) in MEM (Lonza) supplemented with 10% low-endotoxin fetal bovine serum, 1% HEPES, 1% penicillin/streptomycin, and 2% glutamine (complete MEM) per well. Plates were incubated at 37°C and 5% CO₂ for 2 days. To visualize plaques, cells were stained with 1.5% SeaKem agarose in complete MEM containing 1% neutral red (Sigma–Aldrich) per well for 6 h.
Plaque sizes were shown to be associated with the virulence in multiple studies (Takemoto, 1966 (link); Lipton, 1980 (link)). In this study, from photos taken from each group with the same format and size, the diameters of plaques were measured and recorded by the use of ImageJ (National Institutes of Health, Bethesda, MD, USA).

The titers of MNV-1 and TV were determined by plaque assay. Briefly, cells (RAW 264.7 cells for MNV-1 and LLC-MK2 cells for TV) were seeded into six-well plates. On the following day, when the cells were ∼80% confluent, 10-fold dilutions of the samples of unknown virus titer were prepared in complete DMEM, and 1 ml per dilution of the sample was plated onto two wells (0.5 ml per well). The plates were incubated for 1 h at room temperature and manually rocked every 15 min before aspirating the inoculum and overlaying the cells with 1.5% Sea-Plaque agarose (Cambrex, Rockland, ME, United States) in minimum essential Eagle medium (MEME; Lonza) supplemented with 10% low-endotoxin fetal bovine serum, 1% HEPES, 1% penicillin-streptomycin, and 2% glutamine (complete MEME) per well. The plates were incubated at 37°C and 5% CO₂ for 2 days for MNV-1 and 3 days for TV. To visualize the plaques formed by MNV-1, RAW 264.7 cells were stained with 1.5% SeaKem agarose in complete MEME containing 1% neutral red (Sigma- Aldrich, St. Louis, MO, United States) per well for 6 h. To visualize the plaques formed by TV, LLC-MK2 cells were fixed with 3.6% formaldehyde [Sigma-Aldrich; diluted in phosphate-buffered saline (PBS, pH 7.5, Lonza)] for 30 min. The agarose-medium overlays were removed and the cells were stained with 0.1% (w/v) crystal violet (Sigma-Aldrich; diluted in 10% ethanol).