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The manufacturer's protocol was followed and except as noted, incubation was carried out for 1 hour (h) at room temperature (RT).
As noted, incubation of Mtb at pH 4.5 with as little as 3.13 μM BO43 lowered pHIB of Mtb to <5.5 (the limit of detection for the pH-sensitive GFP).
After the noted incubation time, the reactions were quenched by the addition of 1 μg of stop DNA (supercoiled 5 S rDNA) and an equal amount of stop buffer (buffer 1 + 25 mM EDTA), and placed on ice.
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Furthermore, we noted that incubation of young WI-38 HDFs at lower (1%) O2 levels reduced steady-sate levels of the IFI16 mRNA and protein by 50 70% within 24 h of incubation (data not shown).
It should be noted that incubation with a mixture containing anti-αv, -α6 and -β1 integrin mAbs did not totally abolish cell migration in either condition, suggesting the participation of integrin-independent cell adhesion molecules in this process.
Some macroscale reversing effects were noted after incubation at 37°C for one hour.
Bactericidal activity of an extract was noted after incubation for 7 days and an absence of colonies on agar plates.
It should be noted that incubation with ascorbic acid did not decrease the cytotoxicity of compound 12 (not shown).
A diagnosis of OC was made when colony formation was noted during incubation and the presence of pseudo mycelium was shown by Gram staining [ 14].
No matter what the H2S release mechanism for arylthioamides is, it should be noted that incubation of 1 mmol/L of the donors only release H2S with a Cmax value of about 10 µmol/L, which means that the major species in solution is still the donor itself.
[Note: incubation of total RNA with large quantities of DNase I was required to prevent amplification of small genomic DNA sequences during PCR-amplifications using cDNA templates.] Genomewide genotyping was carried out using HumanOmni1-Quad genotype arrays (Illumina) for each of the 52 independent samples in our study (~ 1.14 × 106 genotypes/sample).
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