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In the time course assay (Additional file 1), cells were harvested after 20 h treatment and grown in compound-free medium for 24 h, 5 days and 10 days then placed on chamber slides.
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A potassium sensor was additionally placed on-chip within the analysis chamber to analyze the sample of dialysate.
In order to simulate the occupational exposure scenario, experiments were conducted in a 20.3 m chamber (air exchange rate, 1.0 h− 1) using a 1-compartment model. 2 m of ceramic tiles placed on the chamber floor were sprayed with the SRS product using an airless spray gun identical to the one used in the supermarket.
Four petridishes with water were placed on the chamber floor to additionally humidify the atmosphere.
A rubber o-ring was placed on the chamber surrounding the mounted cornea to facilitate water coupling with the microscope objective in upright configuration.
The inverted cover-slip was placed on the chamber and the outer well was refilled with fresh medium exactly according to manufacturer's instructions.
Cell were placed on special chambers containing 2 ml of recording medium (5% Hanks balanced salt solution, 0.5% glucose, 1% fetal bovine serum, 20 mM Hepes, and 2 mM Glutamax, pH 7.3) and imaged with the DSU system using a 60x 1.42 NA oil immersion objective.
The upper chamber was then placed on the lower chamber of the CIM-Plate which contained 10 nM SDF1.
For deposition of the BCNTs with Si, the samples were placed on the vacuum chamber of a Varian RF sputtering machine.
Briefly, a 5 µl aliquot of prepared sperm sample was placed on a Mackler chamber.
After washing with Krebs' solution, these cells on a coverslip was placed on a recording chamber mounted on the stage of fluorescence microscope (BS50WI, Olympus).
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