Seaplaque agarose

Full-length TBEV RNAs were produced and purified as described above. PS cells were transfected with SP6-transcribed RNAs (1 μg RNA for 1.2 × 10⁵ cells) using Lipofectamine 2000 (Invitrogen) as per the manufacturer’s recommendation. Infectious supernatants were collected at 24 to 72 hpi. Aliquots of virus were diluted with serum-free RPMI 1640 and applied to monolayers of PS cells for 1 h at 37°C. The inoculum was aspirated, and plates were overlaid with RPMI 1640 supplemented with 2% FCS and 1% SeaPlaque Agarose (Cambrex) for 5 days at 37°C for plaque formation. Monolayers were fixed with 4% paraformaldehyde and stained with 0.05% crystal violet. Plaques were counted and virus titers expressed as log₁₀ PFU/mL. All virus work was performed in a Biological Safety Level 3 (BSL3) laboratory. For TBEV growth kinetics, PS cells were infected with WT or NS3/NS5 chimeras at a multiplicity of infection (MOI) of 0.1 for 1 h. Infected cells were washed five times with PBS and incubated with fresh complete medium (2% FCS) at 37°C. Supernatant from infected cells was collected at 0, 4, 8, 12, 16, 20, 24, and 72 hpi and frozen at −80°C prior to further analysis. Experiments were performed in triplicate. Titers of infectious virus at different time points were determined by plaque assay.

Two layer-colony forming assay was carried out as a three dimensional colony growth of human breast carcinoma cells by following the method described previously [10 (link)]. Briefly, 0.5 ml of 0.9% agar (Sea Plaque agarose, Cambrex Bio Science Rockland, Inc. Rockland, ME) in MEM α medium supplemented with 10% FBS was poured into the wells of a 12-well tissue culture plate as a bottom layer. After solidification, 25,000 MDA-231 cells transduced with 50 MOI Ad-ADRP,-FST or-LacZ were suspended in 0.5ml 0.5% agarose in MEM α medium containing 10% FBS and layered over the bottom layer. The cells were incubated at 37°C with 5% CO₂ for growth of colonies. On days 7 and 12, colony growth was evaluated by an automated phase contrast microscope equipped with Micro Analysis Suite (Olympus CKX41, Center Valley, PA). Colonies with an area greater than 5,000 μm² were counted using Micro Suite Five software. The ratio of colony growth of MDA-231 cells was determined between days 7 and 12.

Assays for clonogenic survival of cells were essentially carried out as described by Franken and coworkers [19 (link)]. Briefly, following treatment, 1,000 viable cells (as determined by trypan blue staining) were plated into six-well plates in complete medium without zVAD-fmk, CHX, TRAIL or TNF, cultured for 7 days at 37°C and stained with crystal violet as described above under “viability assays”, except that all steps subsequent to washing with tap water were omitted. Non-adherent U-937 cells were alternatively analyzed for their ability to form colonies in soft agarose by overlaying them onto 2 ml of 0.4% w/v Sea Plaque agarose (Cambrex, East Rutherford, NJ, USA) on top of 3 ml of a 1% w/v peqGOLD agarose underlayer (PeqLab, Erlangen, Germany), both in complete medium. After incubation for 7 days at 37°C, U-937 cells were stained with 1 ml of 3-[4,5-dimethylthiazol-2yl]-2,5-diphenylterazolium bromide (MTT, Sigma, 2.5 mg/ml in PBS) for 2 h at 37°C to allow metabolization of MTT to blue MTT-formazan. Colony formation (>10 cells) was determined from pictures taken with a Lumix DMC-FS10 digital camera (Panasonic, Wiesbaden, Germany).