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The renaturation buffer through the chamber was collected, stirred overnight and then subjected to another round of Ni NTA chromatography as described above followed by desalting with ultra-filtration (6 kD filter).
At different intervals after dextran addition, the medium in the low chamber was collected to detect the transported dextran by liquid scintillation counting.
After voiding, fluid from the uroflow chamber was collected (known as "voided volume," VV).
When the queen died, each colony's inner brood chamber was collected and frozen for subsequent inspection.
Following each culture period, the C. albicans in the upper chamber was collected separately, and cell density was determined by MTT staining, as described above.
To assess migration of nonadherent cells (typically lymphocytes), the medium from the lower chamber was collected, the cells were pelleted by centrifugation, and counting was performed by flow cytometry using Flow-Count beads (Becton-Dickinson, Cowley, UK).
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Cells that had passed through the filter into the lower chamber were collected, and 10 µl of an internal bead control (Bangs Laboratories, Fishers, IN) was added.
Migrated tumor cells on the bottom side of the membranes and in the lower chamber were collected, stained and calculated under a microscope [15].
The cells in the lower chamber were collected and quantified by MPO activity assay.
After 4 h at 37°C, cells that migrated into the lower chamber were collected and counted using a haemocytometer.
After incubation, remaining cells inside the chamber were collected along with cells that have invaded the membrane.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com