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Treated assay plates were incubated with compounds for 72 h and subsequently were developed for endpoint analysis using ATPLite.
The cells were incubated with compounds for 3 days.
The cells grown on the sterile cover slip in 6-well tissue culture plates were treated with compounds for a certain range of treatment time.
Cells were incubated with compounds for 1 h at 37 °C in a humidified atmosphere of 5%% CO2 and 95%% air.
To further assure that compounds are binding with the membrane ergosterol, the cells were incubated with compounds for a definite period of time and amount of unbound compound in supernatant was detected spectrophotometricaly.
The cells were seeded at a concentration of 5 × 104 cell/mL in a volume of 0.8 mL on a sterile cover slip in 6-well tissue culture plates, and were treated with compounds for a certain range of treatment time.
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(b) BMDM was treated with compound for 16 hrs and autophagy induction was determined using Cyto-ID Autophagy Detection kit.
As further evidence that these compounds do not directly bind AR, they showed no target engagement measured by CETSA HT at any point following a time-course experiment in which cells were incubated with compound for a range of timepoints from 10 minutes to 6 hours (Supplementary Fig. S7).
Cells grown on a sterile cover slip in six-well tissue culture plates were treated with compound for a certain range of time.
Genistein was added 8 hours later and cells incubated with compound for 16 hours.
Cells were pre-treated with compound for 1 hour at 37°C then stimulated with IL-6 (200 ng/ml) and IL-6sR (250 ng/ml) for 30 minutes to provide optimal Stat3 activation and nuclear translocation in these cells, as described [31].
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