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In his introduction to the English edition Edward Evans-Pritchard commented on that passage: "Total" is the key word of the Essay.
At each passage, total viable cell numbers were determined as was self-renewal capacity.
In the 200 μM CDCA-treated mES cells cultured for 72 h, Oct4 mRNA expression was downregulated and the phenomenon disappeared at 100 μM CDCA for the second passage (total 144 h incubation with CDCA).
In order to confirm that the expression level of the cre gene was maintained during passage, total RNAs after 1, 7, and 11 passages were also prepared and subjected to northern blotting.
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After clonogenic selection under ethidium bromide, pyruvate, uridine and 72 serial passages, total oxygen consumption and glucose uptake were measured.
In addition, the results of phenotypic analysis showed p75NTR+ cells of different passages (total passages = 30) generated p75NTR+β1-integrin-, p75NTR-β1-integrin and p75NTR-β1-integrin+ progenies, whereas p75NTR- cells generated only p75NTR- cells even in the 30th passage.
At passage 7, total RNA was isolated and miRNA expression measured by microarray.
At passage 7, total RNA was isolated and miRNA expression measured by hybridization-based microarray.
At each passage, the total number of collected cells in the dish was determined.
Cultures were discarded after passage 7. Total RNA was isolated from cultured MEF cell lines using TRIzol® Reagent (Invitrogen, Carlsbad, CA) following manufacturer's instructions, followed by DNase treatment.
Phages were confronted with the naive t0 bacteria and with t1 bacteria, with which they had interacted during the first passage cycle (total number of replicates in assay: 4 phage isolates × 8 replicates × 2 bacteria types = 64).
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