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The surface was regenerated between each experiment with two consecutive injections (0.5 min) of 15 mmol/L NaOH at 30 μL/min followed by a 1 min equilibration phase in the PBS buffer before the subsequent experiment.
The mammary glands were milked over about 5 minutes during a 30 min equilibration phase.
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After equilibration phase (90 min), tissues were exposed to potassium chloride (80 nM) and subsequently phenylephrine (1 μM) to achieve maximal contraction.
Figure 11 illustrates the concentration profile of ACN in a sequence of 10 min alternating gradient runs over the full range of mobile phase composition (HILIC with 100 0% ACN and RP with 0 100% ACN), with 10 min equilibration periods inserted between the alternating gradient runs.
After a 60 min equilibration period, EDV and EDA rings were contracted with phenylephrine.
The total time of the analysis was 30 min, equilibration time 5 min.
Ethanol and acetaldehyde were determined using HS-GC-FID with capillary columns, headspace equilibration temperature (HS-T°) of 70 °C and 20 min equilibration time (HS-EqT).
An additional 25 min equilibration period was then used with the same equilibration buffer to which 2.5% iodoacetamide, instead of 2% DTT, was added.
The mobile phase of 20% acetonitrile in 0.1% aqueous ammonium acetate (w/v) at the flow rate of 1.0 ml min−1 washed the system for 5 min equilibration and continued to another 5 min after injection the sample into the column.
All hearts were perfused for a 15 min equilibration period.
After a 10 min equilibration, VO2 and VCO2 were measured by indirect calorimetry (Vmax29n, Sensormedics) during a 30 min baseline period and during the last 30 min of each four experimental phases to determine net carbohydrate (net CHOox) and net fatty acid oxidation (net FATox) [16].
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