Novel cell separation method for molecular analysis of neuron-astrocyte co-cultures
Front. Cell. Neurosci.. 2014-01-01; 8:
DOI: 10.3389/fncel.2014.00012
Read on PubMed
1. Front Cell Neurosci. 2014 Jan 30;8:12. doi: 10.3389/fncel.2014.00012. eCollection
2014.
Novel cell separation method for molecular analysis of neuron-astrocyte
co-cultures.
Goudriaan A(1), Camargo N(1), Carney KE(2), Oliet SH(3), Smit AB(1), Verheijen
MH(1).
Author information:
(1)Department of Molecular and Cellular Neurobiology, Center for Neurogenomics
and Cognitive Research, Neuroscience Campus Amsterdam, VU University Amsterdam
Amsterdam, Netherlands.
(2)Department of Molecular and Cellular Neurobiology, Center for Neurogenomics
and Cognitive Research, Neuroscience Campus Amsterdam, VU University Amsterdam
Amsterdam, Netherlands ; INSERM U862, Neurocentre Magendie Bordeaux, France ;
Université de Bordeaux Bordeaux, France.
(3)INSERM U862, Neurocentre Magendie Bordeaux, France ; Université de Bordeaux
Bordeaux, France.
Over the last decade, the importance of astrocyte-neuron communication in
neuronal development and synaptic plasticity has become increasingly clear. Since
neuron-astrocyte interactions represent highly dynamic and reciprocal processes,
we hypothesized that many astrocyte genes may be regulated as a consequence of
their interactions with maturing neurons. In order to identify such
neuron-responsive astrocyte genes in vitro, we sought to establish an expedited
technique for separation of neurons from co-cultured astrocytes. Our newly
established method makes use of cold jet, which exploits different adhesion
characteristics of subpopulations of cells (Jirsova etal., 1997), and is rapid,
performed under ice-cold conditions and avoids protease-mediated isolation of
astrocytes or time-consuming centrifugation, yielding intact astrocyte mRNA with
approximately 90% of neuronal RNA removed. Using this purification method, we
executed genome-wide profiling in which RNA derived from astrocyte-only cultures
was compared with astrocyte RNA derived from differentiating neuron-astrocyte
co-cultures. Data analysis determined that many astrocytic mRNAs and biological
processes are regulated by neuronal interaction. Our results validate the cold
jet as an efficient method to separate astrocytes from neurons in co-culture, and
reveals that neurons induce robust gene-expression changes in co-cultured
astrocytes.
DOI: 10.3389/fncel.2014.00012
PMCID: PMC3906515
PMID: 24523672