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At the end of each experiment, cell viability was checked by use of an MTT assay, which was performed as described previously (De Ryck et al. 2014).
After each experiment, cell suspensions were filtered and washed several times with distilled water and filtered cells were dried in oven at 50°C to a constant weight.
The cellular compatibility of the coatings was evaluated through a cytotoxicity experiment, cell proliferation assay, and examination of the adhesion behaviour of osteoblasts.
In the pS2 gene expression experiment, cell monolayers were treated with the carotene concentrate (10−6 M), either with or without supplemented estradiol (10−8 M), and subsequently the RNA was extracted.
In receptor blocking experiment, cell surface Tf receptors were blocked by incubating cells with an excess amount of free Tf for 1 h prior to incubation with Tf-C-SLN and the effect on ROS generation was determined after 24 h treatment (Sahoo and Labhasetwar 2005).
Prior to initiating the experiment, cell cultures were washed 3 times with serum-free medium to remove serum proteins.
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In the hemophilia experiment, cells were taken from the patients, and genes to make a key blood clotting factor were inserted into the cells.
Before the transport experiment, cells were washed three times with warm HBSS (pH 7.4, 37 °C).
In this experiment, cells were starved for 12 h before palmitate treatment.
19 The second analysis involved sequence data from an H3K4me ChIP-Seq experiment (cell-line K562, data available from University of Washington) in which functional loci based on the chromatin signatures can be identified, ie, H3K4me peaks at the promoter of active genes.
In each experiment, cells were harvested separately after 72 h.
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