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After a 72 h migration at 37°C, all cells that had migrated were fixed in 4% paraformaldehyde/PBS and stained with hematoxylin.
Following incubation, cells on the upper side of the chamber were removed using cotton swabs, and cells that had migrated were fixed and stained with Toluidine blue solution.
The filter along with the cells that migrated were fixed and stained with methylene blue.
Stationary cells on the top surface of the membrane were scraped with a cotton swab, and the cells that migrated were fixed with methanol for 30 min.
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Migrating cells were fixed 48 72 h after seeding using 4% PFA for 2 h, permeabilized in 0.5 % (vol/vol) Triton-X 100 for 30 min at room temperature, and stained overnight at 4 °C using Alexa Fluor 488 phalloidin.
Migrating cells were fixed in methanol, stained with hematoxylin and counts averaged at × 20 magnification over five different fields.
Migrating cells were fixed with 20% ethanol and stained with 0.1% crystal violet (Sigma-Aldrich, St . Louis MO, USA).
Migrated HUVECs were fixed, stained with crystal violet, and counted in randomly selected fields under microscopy.
Migrated macrophages were fixed with 4% paraformaldehyde and stained with crystal violet according to the manufacturer's protocol.
Invading or migrating cancer cells were fixed to the lower surface of the transwell membrane with 70%% ethanol, stained with H&E, and counted in five random fields at 200× magnification.
After incubation, cells that did not migrate were scraped from the top compartment, and the cells that migrated through the membrane were fixed and stained using the protocol of the HEMA 3 stain set (Thermo Fisher Scientific, Pittsburgh, PA).
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