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After 48 h of culture, cells were washed twice with phenol red-free and serum-free MEM (Gibco) followed by incubation in 300 µl of the same medium containing vehicle, Bch or E2.
The control cells were replaced with medium containing vehicle only.
Untreated medium containing vehicle DMSO (0.1%) was used as a negative control in this study.
Control cultures were grown in medium containing vehicle (0.1% DMSO) only.
Then, fresh medium containing vehicle or Vidarabine (0 50 μ m) in serum-free or 2% FBS medium was added.
The negative control for all the assays was represented by the untreated medium containing vehicle DMSO (0.1%).
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The percentage of cytotoxicity was calculated using the formula: Cytotoxicity % = A ‒ C min / C max ‒ C min × 100%% A = absorbance measured; Cmin = absorbance of negative control (medium contains vehicle only); Cmax = absorbance of maximum toxicity (medium contains 500 nM staurosporine).
The ice-cold medium was then replaced with pre-warmed medium (37 °C) containing vehicle (control) or 2 μM G-1.
For pharmacological studies, cells were treated in the same manner with graded doses of toxin in medium containing solvent vehicle (control) and with toxin dilutions in medium containing both vehicle and pharmacological agent.
GH3 cells (1.5×105 cells/well, in a 6 well plate) in 2X complete medium (containing either vehicle, ICI 10 nM, or MPP 10 nM) were mixed with 0.3% agar and layered on the bottom gelled layer of agar.
Each of the aortic rings was incubated in 2 mL DMEM medium containing either vehicle (dH2O) or Ang II (100 nmol/L) for 60 min. The inner diameters were calculated by measuring luminal diameter through the transverse section of the thin-vessel ring by using a calibrated micrometer eyepiece.
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