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Cytotoxicity was assessed by analysis of propidium iodide uptake.
Cell viabilty was was determined by subjecting treated or untreated cells to the CellTiter 96 Aqeuous Non-radioactive Cell Proliferation Assay (Promega) or by analysis of propidium iodide stained cells by flow cytometry.
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This was confirmed by the similar DNA content profiles revealed by FACS analysis of propidium iodide stained cells (Fig. S1B ) as well as the similar expression levels of several cell cycle regulators such as p21Cip1 and p27Kip1 (Fig. 1A)In summary, our results suggest that NDRG1 protein levels might not directly impact on cell proliferation in cultured KYSE30 cells.
The effect of radiation exposure (5 gy) on cell cycle distribution was assessed by FACS analysis of propidium iodide stained cells.
In either of these circumstances, the percentage of apoptotic cells was very low, as assessed by FACS analysis of propidium iodide-stained cells.
Cell death and autophagy were quantified by FACS analysis of propidium iodide, Annexin and Lysotracker staining, as well as LC3 translocation.
The cell cycle phase at each time point was determined by FACS analysis of propidium iodide stained cells fixed in 70% ethanol.
We did not observe significant differences in cell growth rates, where A2780 and A2780/GSTP1 cells had doubling times of 0.91 and 1.03 days, respectively (P=0.462), or in cell cycle parameters, assessed by FACS analysis of propidium iodide-labelled untreated cells and stressed cells acutely treated with cisplatin.
Viability of the cells, which was controlled by trypan blue dye exclusion or by quantitative fluorescence analysis of propidium iodide uptake, was > 90%.
Cell death was assessed by determining the integrity of the cell membrane by flow-cytometric analysis of propidium iodide (PI) uptake.
Cellular DNA content was determined by flow cytometric analysis of propidium iodide (PI -labeled cells.
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