title: Enumeration of Neural Stem Cells Using Clonal Assays.
abstract: Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages - astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.
authors: Neural Stem Cell Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR); narayanang1@gis.a-star.edu.sg.
authors: Gunaseelan
authors: G
authors: Narayanan
authors: Neural Stem Cell Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR).
authors: Yuan Hong
authors: YH
authors: Yu
authors: Neural Stem Cell Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR).
authors: Muly
authors: M
authors: Tham
authors: Neural Stem Cell Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR).
authors: Hui Theng
authors: HT
authors: Gan
authors: Neural Stem Cell Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR).
authors: Srinivas
authors: S
authors: Ramasamy
authors: Neural Stem Cell Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR).
authors: Shvetha
authors: S
authors: Sankaran
authors: Neural Stem Cell Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR).
authors: Srivats
authors: S
authors: Hariharan
authors: Neural Stem Cell Laboratory, Institute of Medical Biology, Agency for Science, Technology and Research (A*STAR).
authors: Sohail
authors: S
authors: Ahmed
mesh_terms: D001253:Astrocytes; D002454:Cell Differentiation; D002478:Cells, Cultured; D002999:Clone Cells; D006801:Humans; D058953:Neural Stem Cells; D009474:Neurons; D009836:Oligodendroglia; D013234:Stem Cells