Journal article
Conventional immunomarkers stain a fraction of astrocytes in vitro: A comparison of rat cortical and spinal cord astrocytes in naive and stimulated cultures
JOURNAL OF NEUROSCIENCE RESEARCH, v 99(3), pp 806-826
Mar 2021
PMID: 33295039
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
Astrocytes are responsible for a wide variety of essential functions throughout the central nervous system. The protein markers glial fibrillary acidic protein (GFAP), glutamate aspartate transporter (GLAST), glutamate transporter-1 (GLT-1), glutamine synthetase (GS), 10-formyltetrahydrofolate dehydrogenase (ALDH1L1), and the transcription factor SOX9 are routinely used to label astrocytes in primary rodent cultures. However, GLAST, GLT-1, GS, and SOX9 are also produced by microglia and oligodendrocytes and GFAP, GLAST, GLT-1, and GS production levels are affected by astrocyte phenotypic changes associated with reactive astrogliosis. No group has performed a comprehensive immunocytochemical evaluation to quantify the percentage of cells labeled by these markers in vitro, nor compared changes in staining between cortex- and spinal cord-derived cells in naive and stimulated cultures. Here, we quantified the percentage of cells positively stained for these six markers in astrocyte, microglia, and oligodendrocyte cultures isolated from neonatal rat cortices and spinal cords. Additionally, we incubated the astrocytes with transforming growth factor (TGF)-beta 1 or TGF-beta 3 to determine if the labeling of these markers is altered by these stimuli. We found that only SOX9 in cortical cultures and ALDH1L1 in spinal cord cultures labeled more than 75% of the cells in naive and stimulated astrocyte cultures and stained less than 5% of the cells in microglia and oligodendrocyte cultures. Furthermore, significantly more cortical than spinal cord astrocytes stained for GFAP, GLAST, and ALDH1L1 in naive cultures, whereas significantly more spinal cord than cortical astrocytes stained for GLAST and GS in TGF-beta 1-treated cultures. These findings are important as variability in marker staining may lead to misinterpretation of the astrocyte response in cocultures, migration assays, or engineered disease models.
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Details
- Title
- Conventional immunomarkers stain a fraction of astrocytes in vitro: A comparison of rat cortical and spinal cord astrocytes in naive and stimulated cultures
- Publication Details
- JOURNAL OF NEUROSCIENCE RESEARCH, v 99(3), pp 806-826
- Publisher
- WILEY; HOBOKEN
- Number of pages
- 20
- Grant note
- We thank Neda Bajalo and the Bioresearch Core for their assistance with animal care. Additionally, we thank the following funding sources: NIH R01 [NS092754] to R.J.G., State of New York Spinal Cord Injury Research Program Institutional Grant [C32245GG] to R.J.G, and the National Science Foundation Graduate Research Fellowship Grant No. [DGE-1744655] awarded to J.L. F., [DGE-1744655] awarded to D.L.P., and [DGE-1247271] awarded to A.M. Z. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation.
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Drexel University
- Web of Science ID
- WOS:000596510600001
- Scopus ID
- 2-s2.0-85097303269
- Other Identifier
- 991021860657704721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Neurosciences