To illuminate the inside of the "black-box" neurosphere, we immunocytochemically stained proliferating spheres.
That might account for the kitchen's occasional lack of conviction and the proliferating spheres.
To determine viability of cells, we preincubated spheres for 7 days with different concentrations of BDE-47 or BDE-99 under proliferating conditions followed by further differentiation in presence of PBDEs for 7 days or for 14 days as proliferating spheres.
Here we found in our experiments that although seven of the nine human gliomas gave rise to proliferating tumor spheres, different numbers of spheres arisen from equal primary glioma cells among tumor samples.
Seven primary gliomas of the nine gave rise to proliferating tumor spheres.
In contrast, cells proliferating as spheres from glioblastomas, adult medulloblastoma and ganglioglioma were rapidly lost upon serial passages after 4 to 6 months in accordance with previous studies [ 3, 4].
Typically, if 100,000 cells are plated, greater than 90,000 of the cells die within the first 24 48 hours, leaving 10,000 cells to proliferate, form spheres and ultimately generate about 500,000 cells.
One limitation to our approach is the loss of potentially important antigens from the cell populations that were unable to proliferate under sphere-forming conditions.
Proliferating or differentiating spheres were fixed in 4% paraformaldehyde for 30 min. After washing spheres in phosphate-buffered saline (PBS), they were incubated in a 25% sucrose solution (wt/vol) overnight at 4°C.
This confirmed that RSCs in culture medium as well as in each sphere were proliferating and growing.
In fact, TLX colocalized with KI67, a marker for dividing cells, indicating that TLX is associated with proliferating cells in the spheres.
