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The active site of metalloproteases is composed of a primary sphere of residues co-ordinating the zinc atom, and a secondary sphere of residues that determines proteolytic specificity and activity.
Their primary sphere of influence comes from their role as wives and mothers.
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To assess that the morphological changes are due to the microenvironmental factors, the primary spheres of SUM149PT and SUM1315MO2 were trypsinized and reseeded for the growth of secondary spheres and were observed after 10 days (Fig. 1d).
The polarization ratio at 90° is found to be a monotonic function of primary sphere size and relatively independent of agglomerate size.
From the library of 53 sulfated NSGMs, 11 showed >50% inhibition of primary sphere formation in HT-29 cells without inducing any meaningful inhibition of monolayer growth (see Supporting Information Figure S4 for all results).
A second generation of sarcospheres was formed with an efficiency of 4.7 ± 0.7% (n = 3), yielding a stem cell frequency similar to that of the primary sphere formation assay.
In contrast to the biphasic pattern of the primary sphere formation in irradiated MCF-7 cells, we observed a dose-dependent increase in primary sphere formation in T47D cells, with a two-fold (P = 0.018) maximum increase at the highest dose of 8 × 2 Gy.
The number of secondary spheres arising from a single primary sphere was counted after one week in culture.
Although no obvious difference in primary sphere formation was detected between DSCs and Hep2 cells, the number of spheres formed by DSCs was about two times more than that of Hep2 cells during secondary and tertiary sphere formation.
For secondary sphere formation, primary sphere cultures were filtered using a 70 μm nylon cell strainer, to retain spheres of larger than 70 μm in diameter.
The ability of the cells to form secondary spheres was enhanced compared to primary sphere forming ability, producing on average 454 secondary spheres from 20,000 plated cells (0.055%), compared to the average primary sphere forming ability of 254 spheres from 40,000 plated cells (0.006%).
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