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Chromatography was carried out at a flow rate of 0.5 ml/min using the following gradient; 1 min of 20% solvent B, following by a 3 min gradient increase of solvent B to 40% to elute ET-SO3H and ET. To elute hercynine and S-methyl ET, solvent B was increased to 90% in 1 min, and maintained for 3.5 min before returning to 20% for 3.5 min to re-equilibrate the column.
After 5 min, rats were taken from the cylinder, dried and placed in a warmed place for approximately 15 min before returning to home cages.
Following both pre-test and test sessions, rats were dried with a towel and kept warm for 30 min before returning in their home cage.
The entire [Ca2+]i signal lasted just over 2 min before returning to baseline levels.
These conditions are held for 15 min before returning to the initial conditions.
The gradient was kept at 100% B for 0.5 min before returning to starting conditions.
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Allow to dry before returning the books.
Typically, the solvent gradient was programmed as following: 0 5 min 20% A, 5 15 min 65% A, 15 20 min 100% A, before returning to the initial composition at 30 min. Separated molecules were ionized in the ESI source and analyzed with a Waters Quattro LC triple quadrupole mass spectrometer (Waters, Milford, MA, USA) operating in a Multiple Reaction Monitoring (MRM) scanning mode.
To avoid prolonged application having deleterious effects on slice health, we applied PMA for either 10 min (room temperature) or 5 min (physiological temperature) before returning slices to control solution.
However, the 16 mg/kg dose of Imagent® increased both pulmonary artery pressure (PAP) transiently by an average of 5.7 mmHg (p < 0.05) 2 to 3 min after injection and pulmonary vascular resistance (PVR) by 5.9 mmHg per l/min (p < 0.05) 4 min after injection before returning to preinjection levels.
Wake levels remained in that range for about 5 more min, before gradually returning to baseline.
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