Cell cultures were fixed in 4% paraformaldehyde in PBS or with paraformaldehyde/PBS followed by ice-cold acidic ethanol and HCL for BrdU staining [20 , 22 (link), 33 (link)]. Analyses performed using a confocal laser microscope and computer assisted image analysis (Leica). Quantification of the amount of cells expressing a given marker or marker combinations was determined relative to the total number of DAPI-labeled nuclei or Tuj1+ cells using the Leica lite Software and three-dimensional overlay to avoid false-positive/negative overlay and double counting.
Caspase-3 activity was evaluated as a marker of cell death [19 (link),20 ], using the fluorogenic substrate DEVD-AFC (Ac-Asp-Glu-Val-Asp (DEVD)-pNA (Upstate Biotechnology). Samples were analyzed in a plate reader at 405 nm and enzymatic activity is expressed as arbitrary fluorescent units [20 ,21 ,33 (link)].
Caspase-3 activity was evaluated as a marker of cell death [19 (link),20 ], using the fluorogenic substrate DEVD-AFC (Ac-Asp-Glu-Val-Asp (DEVD)-pNA (Upstate Biotechnology). Samples were analyzed in a plate reader at 405 nm and enzymatic activity is expressed as arbitrary fluorescent units [20 ,21 ,33 (link)].
