Evaluating Cell Proliferation and Migration

MTT and BrdU incorporation assays were performed as previously described [24 (link),14 (link)]. Cells were seeded in 96-well plates at 1 × 10⁴ cells per well. Colony formation was determined by using 3D soft gar matrix with a 0.8% of soft agar (noble agar) base layer followed by 0.4% top agar layer containing cells at a density of 1 × 10⁴ with different treatments in 24-well culture plates. Colonies were counted using inverted light microscopy. For transmigration assay, the ability of migration and invasion of cells was examined using BD BioCoat Matrigel Invasion Chambers (BD Biosciences) according to manufacturer’s instruction. After 24 h, the migrated cells were pelleted and counted using a hematocytometer. To further confirm cell migration, a wound healing assay was performed in 12 well plates (1 × 10⁵ per well). When the cells were grown to 90 to 95% confluences, tranfection of CD133+ cells with siRNA-snoRA42 and scrambled siRNA, as well as mock transfection were performed. Wound lines were created manually by scratching the monolayer with a sterile 200 ml pipette tip and migration of the cells was assessed after 24 hours. Pictures were taken using Nikon inverted phase-contrast microscope (Nikon, Melville, NY). The distance between the parallel lines was measured using ImageJ software. All experiments were carried out at least three times.