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At 175 min of lipid infusion, CEU measurements of forearm muscle MBV were initiated and continued for 40 min as described below.
After 150 min of lipid infusion, paired arterial and venous samples were taken every 10 min three times for measurement of plasma glucose, insulin, FFAs, and lactate.
To avoid interference with the CEU images, no arterial or venous samples were collected from 180 to 210 min of lipid infusion.
Forearm glucose and FFA balances (net uptake or release) were determined from the arteriovenous concentration difference obtained every 10 min from 150 to 300 min of lipid infusion.
Macrophage activation was assayed by using fluorescence microscopy to visualize the phagocytosis of fluorescent beads by differentiated THP-1 macrophages following 30 min of lipid electrophile treatment and removal.
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A central composite design (CCD) was implemented to statistically analyze and evaluate the effect of ultrasonic power (350 750 W) and treatment time (5 30 min) on lipid yield.
Fixed sample was washed with 1% osmic acid for 30 min (for fixation of lipid cell content), washed 3 times with PBS (10 min. for each time), and dehydrated in ascending ethanol concentrations (30, 50, 70, 90, and absolute alcohol) for 30 min for each concentration, then dehydrated sample was infiltrated with acetone for 1 h.
Fluorescent probes were incubated with cells at 37 °C in the dark prior to harvest as follows: (i) 5 μM MitoSOX for 10 min (assessment of mitochondria-generated superoxide), (ii) 25 nM TMRE for 20 30 min (assessment of mitochondrial membrane potential, Δψm), (iii) 5 μM C11-BODIPY581/591 for 30 min (a surrogate marker of lipid peroxidation).
The mixture was centrifuged at 1000 rpm for 10 min and the extent of lipid peroxidation was subsequently monitored by formation of thiobarbituric acid reactive substances (TBARS) as pink chromogen in presence or absence of extracts and standard (L-ascorbic acid).
To isolate highly pure and high yield subcellular compartments, we propose optimal pulse chase paradigm with 15 min of pulse with NH2-lipid-SPMNP at 4 °C or 37 °C.
After 5 min of incubation at 37 °C, lipids were extracted with chloroform:methanol (2 1, v:v).
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