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We examined the silica particles by transmission electron microscopy, and confirmed that they were well-dispersed smooth-surfaced spheres (data not shown).
In the Au-nanoparticle-decorated ZnS spheres (data not shown here), the SEM micrographs provided insufficient information to distinguish the change in surface features of the ZnS spheres following Au decoration.
Primary spheres obtained from CD24− cells could be enzymatically dissociated with trypsin into single cells to give rise to secondary spheres (data not shown).
Staining of three-dimensional spheroids derived from G-28 and U87 cells maintained in non-adherent tissue culture conditions resulted in penetration and uptake of QD-EGF at least 2 or 3 cell layers into the spheres (data not shown).
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However, the growth of the spheres in time (Fig. 1B D) was not due to cell aggregation but was the result of proliferation since plating of gently dissociated glands (clusters of 2 5 cells) in immobilizing semi-solid medium gave rise to sphere formation (data not shown).
NSF-1-containing medium was more efficient than B27-supplemented medium for inducing sphere cells (data not shown).
Indeed, expression of the mesenchymal marker vimentin was up-regulated in SK-sphere cells (data not shown).
In comparison with the low level of ALDH3A1 present in DU145 monolayer cell-derived xenograft tumours (see Discussion for details), higher levels of ALDH3A1 was apparent in DU145 PCSC-produced xenograft tumours, a common observation obtained in all three xenograft tumours produced from either DU145 monolayer or sphere cells (data not shown).
These NSCs also show self-renewal as evidenced by serial sphere passage capacity (data not shown).
Furthermore, human glands also expressed c-Kit exclusively in ductal cells (Fig. S4P,Q), and a subpopulation of these c-Kit+ cells could be isolated from 3 day old spheres by flow cytometry (data not shown).
In addition, a local recurrence tumor also could produce tumor spheres in growth medium (data not shown).
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