Low melting point agarose

Xanthohumol (XN), Aflatoxin B1 (AFB1), dimethylsulphoxide (DMSO), Benzo(a)pyrene (BaP), Minimal Essential Medium Eagle (MEM), NaHCO₃, non-essential amino acids (NEAA), sodium pyruvate ethylenediaminetetraacetic acid (EDTA), NaCl, NaOH, and phenazine methosulfate (PMS) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin/streptomycin, phosphate-buffered saline (PBS), ethanol, fetal bovine serum, and L-glutamine were from PAA Laboratories (Toronto, Canada). Triton X-100 was from Thermo Fisher Scientific (Pittsburgh, PA, USA). Hoechst 33258, trypsin, low melting point agarose (LMP), and normal melting point agarose (NMP) were from Invitrogen (Waltham, MA, USA). The CellTiter96^® AQueous cell proliferation assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTS) was from Promega (Madison, WI, USA). Etoposide (ET) was from Santa Cruz Biotechnology (Dallas, TX, USA). GelRed Nucleic Acid Stain was from Biotium, (Fremont, CA, USA). Tris was from Merck (Darmstadt, Germany). Anti-γH2AX pS139, FITC conjugate human recombinant antibodies were from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). All other reagents were of the purest grade and solutions were made using Milli-Q water. Stock solutions of XN (70 mM) and AFB1 (3.2 mM) for the in vitro studies were prepared in DMSO and stored at −20 °C.

Embryos were mounted in 3% methylcellulose (Sigma) or 1.5% low melting point agarose (Invitrogen) and image captured with a color digital AxioCam HRc camera (Carl Zeiss, Jena, Germany) or SPOT RT3 camera (Diagnostic Inc., Sterling heights, MI, USA). For the confocal images, embryos were embedded with 5% tricaine and images were collected on a Zeiss LSM700 or Nikon Eclipse 90i C1 confocal microscopes, and Z-stacked images (generally 30 slices with 5–10 μm between) were processed with ZEN software (Carl Zeiss) or ImageJ software (NIH, Bethesda, MD, USA).

WT and homozygous zebrafish larvae (scn1Lab^−/− mutants) (6 dpf) were treated as described above. After incubation, larvae were immobilized in 2% low-melting-point agarose (Invitrogen) at room temperature (RT) and the epileptiform activities were measured by noninvasive local field potential (LFP) recording from the skin above the optic tectum (midbrain). The single glass electrode filled with artificial cerebrospinal fluid (ACSF) (124 mM NaCl, 2 mM KCl, 2 mM MgSO₄, 2 mM CaCl₂, 1.25 mM KH₂PO₄, 26 mM NaHCO₃, and 10 mM glucose) was positioned on the skin above the optic tectum. Each recording lasted for 10 min. Epileptiform activity was quantified by using Clampfit 10.2 software (Molecular Devices Corporation, USA60), as reported previously by our group [18 (link), 19 (link)].

PK-15 cells were infected with PRV-GX at a multiplicity of infection (MOI) of 0.1 and harvested at 24 h post-infection. Vero cells were cotransfected with 5 µg PRV-GX genomic DNA and 2.5 µg pUC-IES-EGFP plasmid with Lipofectamine^® 2000 (Invitrogen, Carlsbad, CA, USA). Cell monolayers were treated with DMEM containing 2% FBS and 1% low melting point agarose (Invitrogen, Carlsbad, CA, USA) when the cytopathic effects (CPEs) were observed. Recombinant viruses were screened and purified from the plaques emitting green fluorescence under fluorescence microscopy. After multiple rounds of screening in PK-15 cells, the gI/gE/US9/US2-deleted recombinant viruses expressing EGFP were obtained and named PRV GX-ΔIES-EGFP. Next, Vero cells were cotransfected with the PRV GX-ΔIES-EGFP genomic DNA and plasmid pUC-IES, and a homogeneous viral population of PRV GX-ΔIES, which did not exhibit green fluorescence emission, was screened and purified. This resulting virus mutant was verified by PCR using the primer pair P7F/P7R and sequencing. Then, the TK-deleted recombinant virus was further obtained using methods analogous to those described above based on PRV GX-ΔIES, and the resulting virus mutant PRV GX-ΔTK/IES was verified by PCR using the primer pair P8F/P8R and sequencing.

Vero A cells were seeded in 6-well plates (BD Falcon 6-Well Cell Culture Plate) at a density of 1 × 10⁶ cells/well. The next day, cells were infected with 1 mL of 10-fold serial dilutions of the respective virus variants in assay medium. At 2 h postinfection, the cell monolayers were washed three times with PBS, after which 3 mL of a freshly prepared 1:1 mixture of 1% low-melting-point agarose (Invitrogen, Belgium) and 2× MEM medium (Gibco, Belgium) was added. After 3 d of incubation at 37°C, cells were fixed with 3.7% formaldehyde followed by removal of the agarose overlay and staining with 2% Giemsa staining solution to visualize the virus-induced plaques.