Basic NeuroscienceEnrichment of differentiated hNT neurons and subsequent analysis using flow-cytometry and xCELLigence sensing

- New method to produce highly enriched cultures of differentiated hNT neurons.
- Additional methods to assess the viability and purity of the enriched neuronal cultures.
- xCELLigence measurement of the neuronal net adhesion.
- Method to analyse the cell surface phenotype of neuronal cells using flow cytometry.

BackgroundHuman 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.New methodHere 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.Comparison with existing methodsThis method of enrichment for the neurons is novel and advances the end user applications of the cells.ResultsIn 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.ConclusionsCollectively, 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.