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Periplasmic and cytoplasmic fractions were prepared from cell lysate from the harvest samples using PeriPreps™Periplasting kit (Epicentre, Madison, WI, USA).
Cultured cells were collected at 14 h post-induction, and cell culture medium was collected from the harvest samples by centrifugation at 13,300g for 5 min for analysis and quantification of extracellular ATH35L.
For SDS-PAGE analysis, supernatants from the harvest samples were treated with 4× Bolt® LDS sample buffer (Life Technology, Frederick, MD, USA) which contains both lithium dodecyl sulfate as a denaturing agent and dithiothreitol (DTT) (Life Technology, Frederick, MD, USA) as a reducing agent.
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The histomorphometric analysis of the harvested samples revealed pronounced new bone formation.
The harvested samples of supernatant, obtained from the 4-h co-incubation of effector cells with target cells at an E/T ratio of 20 1, were assessed for the levels of cytokine secretion.
The harvested samples were divided into three groups: group 1 for weighing, group 2 for measuring length, and group 3 for thoroughly rinsed and stored at temperatures below 10 °C in order to assure that highly unsaturated fatty acids (HUFAs) are not metabolized during storage.
The harvested samples were frozen immediately in liquid nitrogen and stored at − 70 °C in a freezer.
The harvested samples were placed in a holding device filled with 70% ethanol and securely closed.
At 5 DAI, the harvested samples of root tips or segments showed prominent swelling, an indication of nematode invasion and establishment.
The harvested samples were frozen in liquid N2, freeze dried and ground to a fine powder and extracted using 80%% ethanol (v/v).
The harvested samples were immediately placed in 10%% formaldehyde fixative, decalcified in ethylene diaminetetracetic acid, dehydrated in increasing concentrations of ethanol, embedded in paraffin and cut sagittally.
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