Seakem le agarose

To determine the viral titer or viral production, LLC-MK₂ or Vero cells were plated at a density that allowed confluence to be reached within 24 h. Monolayers were infected with viruses 10-fold serially diluted in 1 × M-199E medium (GIBCO, Invitrogen) for 2 h at 37 °C with constant agitation following which the infected cells were overlaid with 0.8% Seakem Le agarose (Cambrex, East Rutherford, NJ) coupled with 2X nutrient solution (Earle’s Balanced Salts supplemented with 0.33% (w/v) yeast extract, 0.165% lactalbumin hydrolysate, and 3% FBS for DENV, JEV, and CHIKV or 1.2% Methyl cellulose (Merck KGaA) in 2X DMEM supplemented with 2% FBS for ZIKV. The plates were incubated at 37 °C, 5% CO₂ for 3 (CHIKV), or 6 (DENV, and JEV) days before a second overlay with 0.8% Seakem Le agarose mixed with nutrient containing neutral red and incubated overnight before plaque counting. For ZIKV after incubation at 37 °C, 5% CO₂ for 7 days plates were fixed with 3.7% (v/v) formaldehyde (Merck KGaA) in 1X Phosphate Buffer Saline (PBS) for 2 h, and stained with 1% crystal violet dye prior to plaque counting. The plaques were counted and viral titer was determined in terms of plaque forming unit/ml (pfu/ml).

DNeasy plant mini kit (Qiagen, Hilden, Germany) was chosen as a suitable procedure for DNA extraction based on the results of our previous study [33 (link)]. Genomic DNAs were isolated and purified from 100 mg flour. The purity and concentration of the extracted DNAs were estimated by spectrophotometer DeNovix DS-11 (DeNovix Inc. Wilmington, NC, USA). The quantity and integrity of DNA were evaluated using electrophoresis (VWR International, Radnor, PA, USA) on 1% agarose gel (SeaKem LE agarose; Cambrex, East Rutherford, NJ, USA) containing 1 μg/mL of ethidium bromide (Sigma-Aldrich, St. Louis, MI, USA). The agarose gels were visualized under ultraviolet (UV) light and a digital image was obtained using a gel documentation system PhotoDoc- It imaging system (UVP, Upland, CA, USA).

Nodules of Cea. thyrsiflorus were harvested 8 months after infection with Cv1. Several nonlignified tips of nodule lobes were cut off and fixed in 3% paraformaldehyde, 0.1% Tween-20, 0.1% Triton X-100 in 10 mM phosphate buffer pH 7.2 overnight before being washed and dehydrated in a graded EtOH series until 70% EtOH and stored at room temperature. Later, they were rehydrated in a graded EtOH series and embedded in 2% agarose (SeaKem LE agarose, Cambrex, Karlskoga, Sweden). Longitudinal sections (45 µm) were prepared on an vibrating blade microtome HM 650 V (Microm, Walldorf, Germany), stained with 0.001% 4′,6-diamidino-2-phenylindole (DAPI) for 30 min and analyzed under a confocal laser scanning microscope LSM 780 (Carl Zeiss, Jena, Germany).

DNA was analyzed by agarose gel electrophoresis using 1, 1.5, and 2% agarose gel (SeaKem LE agarose, Cambrex, gels for genomic and amplified DNA). Electrophoresis was performed using 1× Tris–Borate EDTA (TBE) buffer containing 1 μg/mL of ethidium bromide (EtBr) and a constant voltage of 100 V for 50 min. The DNA bands were visualized and images were acquired using Gel Doc XR+ Imaging system (Bio-Rad Laboratories Inc., Germany).

SeaKem^® LE Agarose was purchased from Cambrex Bio Science Baltimore Inc. (Baltimore, MD, USA) and dissolved at a concentration of 5% (w/v) in purified water. The solution was heated to a boiling temperature to ensure complete dissolution and then poured into Petri dishes to a final height of 3 mm (145 × 20 mm, Greiner Bio-One, Kremsmünster, Austria). The solution was allowed to cool down for 1 h to solidify and used for model system fabrication as described below and stored in phosphate-buffered saline (PBS) for 1 day before testing. Another agarose solution was produced at a concentration of 0.5% (w/v) and used as a soft biomaterial as described below.

Vero (ZIKV) or LLC-MK₂ (DENV and JEV) cells were cultured in 6-well cell culture plates at 37 ^ºC with 5% CO₂ for 22–24 h to obtain approximately 90% confluency. Before infection, cell culture supernatant (control) or stock virus were 10-fold serially diluted with BA-1 virus diluent (1× medium 199/Earle’s balanced salts, 0.05 M Tris-HCl [pH 7.6], 1% bovine serum albumin fraction V, 0.075% NaHCO3, 100 U of penicillin-streptomycin per ml). Cells were infected with 200 µl of the diluted cell culture supernatant or diluted virus at 37 ^ºC for 2 h with gentle rocking every 10 min. After that, the infected cells were overlaid with 4 ml of overlay medium (1.2% methylcellulose (Merck KGaA, Darmstadt, Germany) in DMEM supplemented with 2% FBS for ZIKV, or 4 ml of 0.8% Seakem LE agarose (Cambrex Bio Science Walkersville, Inc., Walkersville MD) in 1X nutrient solution for DENV 2 and JEV). The infected cells were incubated for a further 7 days at 37 ^ºC with 5% CO₂. On day 7, the overlay medium was removed and infected cells were then fixed with 1 ml of 3.7% formaldehyde (Merck KGaA, Darmstadt, Germany) in PBS for at least an hour at room temperature. After that, the formaldehyde was removed and the cells were washed with water prior to staining cells with 1% crystal violet (Merck KGaA, Darmstadt, Germany) in 20% ethanol. All samples were assayed in duplicate.