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To determine the minimum bactericidal concentration (MBC), aliquots from clear wells containing increasing concentrations of the compound were plated onto antibiotic-free agar plates, and the lowest concentration at which no visible growth was observed was reported as the MBC.
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The compounds were plated at five different concentrations in 10-fold dilutions covering a 10,000-fold concentration range (e.g., 1 10,000 nM).
24 hours before the addition of the tested compounds, cells were plated in 96-well plates (Sarstedt, Newton, NC, USA) at a density of 2 × 10 cells per well.
For compound treatment, cells were plated in 96-well plates at a density of 1.0 × 105 cells/mL and allowed grow to 100% confluence over two days.
For compound treatment, the cells were plated at the same density as the siRNA transfection, and the compounds were diluted in OPTI-MEM to 10× concentrations and added 1 2 days later.
For initial screening 2 ul of test compounds resuspended in DMSO were plated in triplicate wells in a black opaque Optiplate (Perkin Elmer) at a concentration of 50 mM to achieve a final concentration of 3.33 mM when resuspended in 30 uL of reaction solution.
For FRAP of compound mutants (Fig. 6), cells were plated and transfected as above, and imaged using a Nikon TE2000 microscope with a C1Si confocal using the 60× oil immersion objective.
The compounds were removed and 5×104 cells were plated and incubated for 14 days [23].
After determination of the optimal and safe concentrations of dietary compounds, approximately 8 × 10 cells/2 ml were plated in 6-well plates.
Multiscreen acceptor filter plates containing compound were then coupled to the receiver plates and incubated at room temperature for 20 h.
Butanolic extract and the purified compound were subjected to both spot and plate incorporation methods of Ames test to evaluate their mutagenicity (Maron and Ames 1983).
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