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The cell number in the culture was determined by absorbance measurements (CellTiter 96 Aqueous One Solution Reagent, Promega) as described by the manufacturer.
Sample quadruplicates were incubated at 37°C for 4 min. Unoxidized Fe2+ was reacted with 60 µl of chromogen solution (reagent C) and absorbance was measured at 600 nm with Smart Spec Plus (BioRad) spectrophotometer.
Subsequently, each well was added 20 µl of one solution reagent using CellTiter 96 AQueous One solution Cell Proliferation Assay kit (Promega, Madison, WI, USA), and incubated for 3 h.
20 µl of the CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI) were added into each well of the 96 wells plate containing cells in 100 µl culture medium, incubated with the reagent for 3 hours at 37°C in 5% CO2 and absorbance at 490 nm was recorded.
Lung tissue was homogenized and suspended in a potassium phosphate (50mmol/l) buffer solution (reagent 1).
CellTiter 96 AQueous One Solution Reagent (Promega) was added to each well following the manufacturer's instructions.
CellTiter 96 Aqueous One Solution reagent was from Promega UK, Southampton, UK.
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Slides were mounted with a mounting-solution reagent containing DAPI (Vector Lab, Burlingame, CA, USA) and then analyzed using a Zeiss Axiovert 200 fluorescence microscope with AXIOVISION 4.2 software (Carl Zeiss, Jena, Germany).
After treatment, 20 μl of CellTiter 96® AQueous One Solution Reagent were dropped into each well of plates.
The CellTiter 96 AQueous One Solution Reagent (Promega, Madison, WI, USA) was used in accordance with manufacturer's instructions.
Cell proliferation was determined by a modified MTS assay with CellTiter 96® AQueous One Solution Reagent (Promega, Madison, WI).
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