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Cell content: Cells can handle almost any data.
To measure the cellular ATP content, cells were reverse transfected with siRNAs in a 96-well plate at 1000 cells per well.
For flow cytometry (FCM) analysis of the DNA content, cells were treated, collected, and fixed in 70% ethanol for at least 24 h.
To analyze DNA content, cells were fixed in ice-cold 70% ethanol spun down and resuspended in 50 mM sodium citrate buffer pH 7.0.
In addition, insect cells (C6/36), previously grown in medium with cholesterol, were assayed as a cellular control of low cholesterol content cells.
To analyze DNA content, cells were trypsinized, washed twice with PBS, then were suspend in 75% chilled ethanol and kept at 4°C over night.
To measure the intracellular glycerol content, cells were collected by centrifugation, washed with distilled water and then extracted by boiling at 100°C for 10 minutes.
To determine budding indices and DNA content, cells in aliquots taken at each time point were pelleted by centrifugation and resuspended in water (for determining budding index) or 70% ethanol (for flow cytometry).
To measure the sub-G1 DNA content, cells were fixed in ice-cold ethanol (70%), re-suspended in phosphate buffered saline containing 40 µg/ml RNase and 20 µg/ml propidium iodide and then subjected to FACS analyses.
Cells in G0 (G0CD34+) were identified by their 2n DNA and minimal RNA content; cells in G1 (G1CD34+) were defined as those with 2n DNA and high RNA staining [28].
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Cells treated with NSC23766 alone in the absence of IR showed no increase in amount of G2/M DNA-content cells at all time points tested (open square).
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