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Astrocytes were grown for 2 weeks in low glucose and then treated with tunicamycin for 16 h and dye transfer was assayed.
After cultured astrocytes were treated for the time intervals indicated, LYVS transfer was assayed by impaling a single astrocyte with a micropipette, allowing the dye to diffuse for 2 min, and then the dye-labelled area was measured using MetaVue software.
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Conjugative transfer of resistance genes was assayed in MH broth using E. coli J53 (auxotropic for proline and methionine and resistant to rifampin and nalidixic acid) as a recipient and an initial donor/recipient ratio of 0.1.
(B ) Label transfer from tRNA-ProUGG mutants was assayed with Ssa2p.
The amount of dye retained is a measure of the number of cells that invaded through the collagen layer and was assayed by transferring samples to 96-well plate and reading optical density on a TECAN GENios Microplate Reader at 560 nm.
The released phosphate was assayed by transferring 60 μL of each reaction mixture to two separate wells on a new transparent 96-well microtiter plate (Greiner Bio-One, Kremsmünster, Austria) and by adding 120 μL of freshly prepared stopping solution of 0.375 M H2SO4, 0.75% (w/v) (NH2 4MoO4 · 4H2O, 0.7% (w/v) SDS and 3% (w/v) FeSO4 · 7H2O to each well.
This was also the case when serum samples from an additional set of similar SCID transfer groups were assayed 67 days after the first cell transfer, indicating that the appearance of PG-specific Abs in serum was not simply delayed in the T cell-depleted transfer recipients (data not shown).
Of the other unc-18 mutants, only two, md1401 and md1264, move sufficiently well to allow sperm transfer to be assayed.
When testing animals specifically for sperm-transfer initiation, animals were assayed until the first tonic spicule insertion and transfer was noted.
To observe the effect of mutations Y143C and Y143R on IN activity and RAL sensitivity, the mutated integrases were produced, purified and in vitro 3'end processing, strand transfer and concerted integration were assayed according to standard procedures previously used in our laboratory [16], [21].
Dye transfer was also assayed in astrocytes cultured in media containing 5.5 or 25 mmol/l glucose for up to 21 days in the presence or absence of compounds to reduce ROS/RNS levels or that facilitate protein folding.
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