Anti cd133 1 pe ac133 clone

Actin fibers and the nucleus were stained after 24 h of treatments. Cells grown on cover glass were washed with DPBS (Welgene, Korea), fixed with 4% paraformaldehyde (Gentic, USA) for 20 min, and permeabilized by 0.1% of Triton X-100 (Sigma-Aldrich, Korea) in PBS for 25 min at room temperature. After washing with DPBS, actin fibers were stained by phalloidin dye (5 units/ml, Phallotoxins; Invitrogen) for 30 min at room temperature. After washing thrice with DPBS, the samples were mounted with the Prolong gold anti-fade reagent with DAPI (Molecular probes; Invitrogen) and observed under fluorescence microscope (Ti-U, Nikon).⁴² For CD133 expression, after dissociation with accutase, gliospheres cells were stained with anti-CD133/1-PE (AC133 clone; Miltenyi Biotec, USA)⁴² and further processed as described above.

Briefly, post treatment of sphere-cultured glioma cells with metallacycle, sphere size was monitored at day 1 and 6 by using Motic Images Plus 2.0 in three randomly chosen fields. For clonal assay, glioma sphere cells were plated into 96-well plates at a density of 1 individual cell each well. After 1 day, each well was visually checked for the presence of a single cell. The clones were grownup and clone formation was monitored at 1 and 6 days. Numbers of clones were counted under microscope [42 (link)]. For self-renewal assay, individual cell was seeded in each well of 96 well plate and the size of sphere was measured under phase-contrast microscope [42 (link)]. For CD133 expression, after dissociation with accutase, gliospheres cells (5 × 10⁵) were labeled with anti-CD133/1-PE (AC133 clone; Miltenyi Biotec, Auburn, CA, USA). For each sample the respective control was prepared without antibody staining. All samples were incubated for 20 minutes at 4°C, washed twice with PBS and immediately analyzed using a BD FACSVerse cytometer and the FACS suite software [42 (link)].