Fluorescent activated cell sorting (FACS): a rapid and reliable method to estimate the number of neurons in a mixed population.
Cells derived from the central nervous system (CNS) are usually characterised by manual counting on slides after specific immunolabelling. In this study, we investigated the possibility of using flow cytometry to determine the proportion of neurons, astrocytes or microglial cells in primary cultures. We show that parameters other than physical features are necessary to discriminate between these different cell types because of some overlap in their size and granulosity. We then used specific antibodies against intracellular markers such as Tuj-1 or GFAP to discriminate neurons from astrocytes by flow cytometry. The labelling was specific and reliable, allowing quantitative studies. Indeed, we did not find any significant difference in the number of Tuj-1 and GFAP-positive cells in primary cultures of neuronal and glial cells as determined by manual counting on slides or flow cytometry. More importantly, similar data were obtained in mixed populations, indicating that flow cytometry can be used for quantitative studies of heterogeneous cultures. The flow cytometry therefore appears to be a reliable method for the phenotypic characterisation of CNS-derived cells. This technique which enables a rapid analysis of numerous samples, might be particularly interesting for the study of neural stem cell differentiation.
Sergent-Tanguy
Solène
S
INSERM U437, 30 bd Jean Monnet, 44093 Nantes Cedex 01, France.
Chagneau
Carine
C
Neveu
Isabelle
I
Naveilhan
Philippe
P
D000818:Animals; D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D001253:Astrocytes / Q000166:cytology; D030161:Avian Proteins; D051926:Basigin; D001798:Blood Proteins; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D048429:Cell Size; D002478:Cells, Cultured; D018920:Coculture Techniques; D005110:Extracellular Space / Q000378:metabolism; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D008562:Membrane Glycoproteins / Q000378:metabolism; D016900:Neurofilament Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D015203:Reproducibility of Results; D013997:Time Factors; D014404:Tubulin / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism
D015703:Antigens, CD; D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D030161:Avian Proteins; D001798:Blood Proteins; C102624:Bsg protein, Gallus gallus; C079432:Bsg protein, rat; D005904:Glial Fibrillary Acidic Protein; D008562:Membrane Glycoproteins; D016900:Neurofilament Proteins; D011506:Proteins; D014404:Tubulin; D014746:Vimentin; C066858:neurofilament protein NF 68; C051774:neurofilament protein H; D051926:Basigin