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Cells were grown in standard culture conditions until confluence, after which time the medium was replaced with serum free medium to eliminate exogenous free fatty acids, notably FBS mediated 18∶1 delivery.
Transformed yeast cells were grown for 24 h at 30°C with shaking in SDCAA medium (yeast nitrogen-based casamino acid medium containing 20 glucoselucose) and passed one time into fresh medium to eliminate dead cells.
Prior to the 6-h induction with 2% galactose, each starter culture was incubated in SC ura- 2% raffinose medium to eliminate any traces of glucose that might inhibit the GAL promoter expression.
Before plating, cultured B cells were washed 5 times with complete medium to eliminate the Ig present in the supernatants.
Conditioned media were obtained using FBS-free culture medium to eliminate the endogenous proMMPs found in FBS.
Infected cells were incubated at 37°C, 5% CO2 for 4 h and then washed three times with fresh RPMI 1640 medium to eliminate extracellular bacteria.
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After two days, medium was changed to eliminate non adherent cells and cultivated in fresh medium for five days.
When a model lettuce agar medium was used to eliminate possible interactions with competing flora the direct effects of the atmosphere enhancing the growth of L. monocytogenes was also observed.
To obtain a quantitative estimate of the number of internalized bacteria, HT29 cells were incubated for further 3 h and 6 h with gentamicin (20 µg/mL) added to the medium in order to eliminate extracellular bacteria.
Each culture was twice diluted into fresh medium during growth to eliminate unlabeled biomass.
BIO was removed from the secondary passage medium in order to eliminate the confounding effect of BIO on cell proliferation.
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