Discrimination of cell types in mixed cortical culture using calcium imaging: A comparison to immunocytochemical labeling

Mark Pickering, Brian W. Pickering, Keith J. Murphy, John J. O'Connor

Research output: Contribution to journalArticlepeer-review

16 Scopus citations

Abstract

Neuronal-glial interactions in the central nervous system are important for both normal function and response to pathological states. Differences in calcium processing between these cell types may be exploited to allow dynamic differentiation using calcium-imaging protocols without the need to fix and immunostain the study population. Mixed rat primary cortical cultures were grown on coverslips, incubated for 30 min in 2 μM fluo-3 AM and mounted in a devised, low volume imaging chamber. Calcium influx was measured over the duration of a 50 s exposure to 30 μM glutamate in all cells. Cells were then fixed in situ, and immunostained for NeuN and GFAP. Direct comparison between live calcium dynamics and cell type markers were made. Over the duration of the glutamate exposure, those cells that subsequently stained for NeuN exhibited a sustained increase in intracellular calcium, whereas GFAP positive and non-staining cells exhibited a decline over the duration of the glutamate exposure. We found that examining the average calcium fluorescence over the last 10 s of glutamate exposure allowed the identification of cells as neuronal if the average was >85% of the maximal calcium change, or non-neuronal if the average was <85% of the maximal calcium change. This technique compares very favourably to the established technique of immunocytochemical labeling for the identification of cell types; both techniques agreed in their classification of cells as neuronal or non-neuronal 96.83% of the time. However, this technique cannot reliably distinguish between non-neuronal cell types.

Original languageEnglish (US)
Pages (from-to)27-33
Number of pages7
JournalJournal of Neuroscience Methods
Volume173
Issue number1
DOIs
StatePublished - Aug 15 2008

Keywords

  • Calcium imaging
  • Immunocytochemistry
  • Primary culture

ASJC Scopus subject areas

  • Neuroscience(all)

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