title: Evaluation of proliferation of neural stem cells in vitro and in vivo.
abstract: This unit describes two basic protocols for the detection of the proliferation of neural stem cells (NSC). The first one addresses cell proliferation in cultures, starting with primary cell cultures isolated from the mouse subventricular zone (SVZ), in which SVZ-derived NSC are kept in culture as neurospheres. By using this culture system, we are able to study different stages of adult neurogenesis, such as proliferation, differentiation, migration, and survival. Thus, in the first basic protocol, we describe two different techniques to evaluate cell proliferation based on EdU incorporation: (a) immunocytochemistry and (b) flow cytometry. EdU, a new thymidine analog, which is detected by a reproducible and sensitive method based on click chemistry, does not require DNA denaturation, as is the case with BrdU. Thus, co-labeling of EdU with other specific antibodies of extracellular or intracellular targets, as well as other DNA dyes, is possible. In the second basic protocol, we describe an in vivo assay to evaluate proliferation of NSC in the dentate gyrus of hippocampus of adult mice, by both BrdU and EdU detection. With this approach, it is also possible to study different stages of adult neurogenesis, by co-labeling thymidine analogs with other specific markers, such as doublecortin (DCX) or neuronal nuclei protein (NeuN).
authors: Centre for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal.
authors: Maria Inês
authors: MI
authors: Morte
authors: Bruno P
authors: BP
authors: Carreira
authors: Vanessa
authors: V
authors: Machado
authors: Anália
authors: A
authors: Carmo
authors: Isabel
authors: I
authors: Nunes-Correia
authors: Caetana M
authors: CM
authors: Carvalho
authors: Inês M
authors: IM
authors: Araújo
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002552:Cerebral Ventricles / Q000166:cytology; D000089662:Doublecortin Protein; D005434:Flow Cytometry; D005557:Formaldehyde; D005780:Gelatin / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011108:Polymers; D016707:Tissue Fixation
chemical_list: C000714847:Dcx protein, mouse; D000089662:Doublecortin Protein; D011108:Polymers; D005557:Formaldehyde; D005780:Gelatin; C003043:paraform