Biotool tunel apo green detection kit

Primary isolated NCSC-like cells were cultured as spheres in 6-well plates for 14 days without adding new media, after which the cell suspensions were seeded in 96-well plates. CellTiter 96® AQueous MTS reagent (Promega) was added at 4 mg/ml to each well, and cells were incubated for 4 h at 37°C. The absorbance of each well was measured using an EL800 Microplate Reader (BioTek) at 490 nm. The ability for self-renewal was measured by secondary sphere formation assay. To obtain single cells, spheres were incubated in 1 mg/ml collagenase type I (Sigma) and 1 mg/ml collagenase type IV (Life Technologies) for 5 min, then mechanically dissociated by pipetting. Single cells were seeded into 96-well plates (2000 cells per well) in StemPro®. After 14 days, the relative growth of spheres in each well was measured using CellTiter 96® AQueous MTS reagent. Cell death was assessed with the red cell-impermeant viability indicator EthD-1 (Life Technologies). Dead cells, marked by bright red to orange fluorescence, were observed using a Nikon TE2000 inverted microscope (Nikon Instruments) using 10, 20 or 40x objective lenses and photo capture was performed with Q-Imaging Retiga EX digital camera. TUNEL assay was performed on sectioned skin reconstructs according to the manufacturer’s protocol using the Biotool^™ TUNEL Apo-Green Detection Kit (Selleck Chemicals, Houston, TX).