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The approach is improved by a new algorithm of waviness envelope construction and a morphologic filter using the waviness envelope as a low-frequency surface of filtering.
Others also observed a reduction of the surface of filtering blebs and their flattening after removal of cataract in the eyes previously subjected to trabeculectomy.
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Addition of exogenous PtdIns 3,4,5 P3 to the apical surface of filter-grown MDCK cells causes the relocalization of basolateral proteins to the apical surface and the removal of apical proteins from the apical surface [49].
Once rehydrated, 2.5×104 cells were resuspended in the RPMI (500 µl) containing 1% FBS with ±10−8 M E2 ±10−6 M fulvestrant ±25 µM DIM and were carefully transferred onto the upper surface of filters in the chamber.
Invading cells on the bottom surface of filter were fixed in methanol and stained with Giemsa.
The nonmigrated cells on the upper surface of filter were removed by cotton swab.
Migration values were expressed as means ± S.D. of the number of migrated cells × 100%/total cells counted on the lower surface of filter.
After a 6-h incubation at 37°C, cells (2.5 × 10, 0.2 ml−1) on the upper surface of filters were scraped; filters, were then fixed in methanol, stained with Wright-Giemsa and photographed as above.
Cells that migrated to the lower surface of filters were detected with traditional H&E staining, and five fields of each well were counted after 4 24 h of incubation at 37°C with 5% CO2.
Following 48 h of incubation, migrated or invasive cells on the lower surface of filters were fixed and stained with the Diff-Quik stain (Sysmex, Kobe, Japan), and stained cell nuclei were counted directly in triplicate.
After incubating at 37°C and 5% CO2 for 4 h, the migrated cells on the lower surface of filter were fixed with 4% formaldehyde at RT for 15 min and stained with Hocechst at RT for 15 min.
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