title: Enrichment of differentiated hNT neurons and subsequent analysis using flow-cytometry and xCELLigence sensing.
abstract: BACKGROUND
  Human neurons (hNT neurons), obtained from the NTera2/D1 precursor cell line, are highly valued by many neuroscientists as isolation of adult human primary neuronal cells continues to elude us. hNT neurons are generated by differentiation of the NT2 precursors for a period of 4 weeks followed by 2 weeks of mitotic inhibition. This yields a heterogeneous population of neuronal phenotypes and underlying astrocyte precursors, the latter of which are very difficult to visualise using standard light microscopy. Such a mixed culture is acceptable for some applications (e.g. measurement of synaptic plasticity), whereas others (e.g. proteomics or transcriptomics) require almost pure cultures of hNT neurons.
  
  
  METHODS
  Here we describe a simple method for obtaining highly enriched cultures of hNT neurons following the first neuronal harvest and detail several additional methods, namely flow-cytometry and xCELLigence© biosensor technology, to rapidly and reliably determine the purity and viability of the cultures.
  
  
  METHODS
  This method of enrichment for the neurons is novel and advances the end user applications of the cells.
  
  
  RESULTS
  In addition, we apply the enrichment method to conduct analysis of cell-surface markers using flow-cytometry on the enriched neuronal cells. Furthermore, we apply this method to generate enriched neuronal cells on which we conduct analysis of cell-surface markers using flow-cytometry.
  
  
  CONCLUSIONS
  Collectively, this paper describes several new advances, which will create opportunities when using these cells and similar preparations, and provides the protocol for analysis of these cells using flow-cytometry and biosensor technology.
authors: Department of Pharmacology and Centre for Brain Research, School of Medical Sciences, The University of Auckland, Faculty of Medical and Health Sciences, Private Bag 92019, Auckland 1142, New Zealand; Department of Engineering Science, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors: Christa
authors: C
authors: MacDonald
authors: Department of Engineering Science, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand.
authors: Charles P
authors: CP
authors: Unsworth
authors: Department of Pharmacology and Centre for Brain Research, School of Medical Sciences, The University of Auckland, Faculty of Medical and Health Sciences, Private Bag 92019, Auckland 1142, New Zealand. Electronic address: s.graham@auckland.ac.nz.
authors: E Scott
authors: ES
authors: Graham
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002454:Cell Differentiation; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology / Q000502:physiology; D055495:Neurogenesis; D009457:Neuroglia / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D013997:Time Factors
chemical_list: D015703:Antigens, CD; D009419:Nerve Tissue Proteins
keywords: Enriched; Flow-cytometry; Neurons; Purity; hNT; xCELLigence