Conventional immunomarkers stain a fraction of astrocytes in vitro: A comparison of rat cortical and spinal cord astrocytes in naive and stimulated cultures
Conventional immunomarkers stain a fraction of astrocytes in vitro: A comparison of rat cortical and spinal cord astrocytes in naive and stimulated cultures
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DOI：
10.1002/jnr.24759
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发表时间：
2020-12-08
影响因子：
4.2
通讯作者：
Gilbert, Ryan J.
中科院分区：
文献类型：
--
作者：
Balouch, Bailey;Funnell, Jessica L.;Gilbert, Ryan J.
星形胶质细胞在整个中枢神经系统中负责各种各样的基本功能。蛋白标记物胶质细胞酸性蛋白（GFAP）、谷氨酸天冬氨酸转运蛋白（GLAST）、谷氨酸转运蛋白-1（GLT-1）、谷氨酰胺合成酶（GS）、10-甲酰四氢叶酸脱氢酶（ALDH 1 L1）和转录因子SOX 9通常用于标记原代啮齿动物培养物中的星形胶质细胞。然而，GLAST、GLT-1、GS和SOX 9也由小胶质细胞和少突胶质细胞产生，并且GFAP、GLAST、GLT-1和GS产生水平受到与反应性星形胶质细胞增生相关的星形胶质细胞表型变化的影响。没有一个小组进行了全面的免疫细胞化学评价，以量化这些标记物在体外标记的细胞的百分比，也没有比较在幼稚和刺激培养的皮质和脊髓来源的细胞之间的染色变化。在这里，我们量化了从新生大鼠皮层和脊髓中分离的星形胶质细胞、小胶质细胞和少突胶质细胞培养物中这六种标记物阳性染色的细胞百分比。此外，我们将星形胶质细胞与转化生长因子（TGF）-β 1或TGF-β 3孵育，以确定这些标记物的标记是否被这些刺激改变。我们发现，只有皮质培养物中的SOX 9和脊髓培养物中的ALDH 1 L1标记了幼稚和刺激的星形胶质细胞培养物中超过75%的细胞，并染色了小胶质细胞和少突胶质细胞培养物中不到5%的细胞。此外，显着更多的皮质比脊髓星形胶质细胞染色的GFAP，GLAST，和ALDH 1 L1在幼稚的文化，而显着更多的脊髓比皮质星形胶质细胞染色的GLAST和GS在TGF-β 1处理的文化。这些发现很重要，因为标记物染色的变异性可能导致共培养、迁移试验或工程疾病模型中星形胶质细胞反应的误解。
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.