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Cells were then analyzed using the Becton Dickinson Facscan (BD bioscience) and levels of WNV positive cells were quantified by reporting the proportion of cells with fluorescent signal exceeding cells derived from a negative control well inoculated with media only.
BAC clones maintained in 384-well microplates were preinoculated in 384-well microplates containing 70 μl 2× LB medium plus chloramphenicol (12.5 mg/ml) per well, inoculated in 96-well plates containing 1.2 ml of the same medium per well, and grown at 37°C with shaking at 250 rpm for 10-15 h.
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After 24 h, planktonic growth was observed in all wells inoculated with bacteria, while no growth was observed in any negative control wells containing media alone (b).
Each experimental section included the following controls: wells of uninfected macrophages; wells inoculated with bacteria only.
A significant reduction in cytopathology was observed in wells inoculated with these viruses and antiserum to EHDV-6 (CSIRO 753).
This was further demonstrated by the fact that the fresh weights and chlorophyll concentrations of the wells inoculated with the recombinant strains were higher than that of the PAO1 and pMEKm12/PAO1 vector control strains (Figure 3B).
After 7 days, about 72.6±1.28% of AGS-Wnt1 wells produced spherical colonies compared with only 36.3±0.98% of wells inoculated with N-Control cells.
Turbidities in wells inoculated with P. inflatum PT52 were the highest for these compounds, supporting its better growth on carbohydrates compared to the other species.
The number of plaques was then determined and activity calculated based on the reduction in average number of plaques, in wells where the compound was pre-incubated with the virus, compared to the control wells inoculated with untreated virus.
Next, the cultures were diluted 1 100, and 200 µL of the diluted cultures, per well, were inoculated into 96-well polystyrene microtiter plates.
Cells (4 × 10 or 5 × 10 per well) were inoculated in a 96-well plate and treated with CY or FCY for 24 or 48 h.
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