Conventional immunomarkers stain a fraction of astrocytes in vitro: A comparison of rat cortical and spinal cord astrocytes in naïve and stimulated cultures
In the present study, we analyzed the percent immunopositive cells in astrocyte-enriched, microglia-enriched, and oligodendrocyte-enriched cultures isolated from the cortex or spinal cord of neonatal rats for six markers that have been previously used to label astrocytes: GFAP, GLAST, GLT-1, GS, ALDH1L1, and SOX9. We also stimulated astrocyte cultures with either transforming growth factor (TGF)-β1, shown to induce reactive astrogliosis in rodent cortical astrocytes in vitro, or TGF-β3, shown to dampen reactivity markers in astrocytes in vitro. Markers were evaluated based on the following criteria: (1) ability to stain all astrocytes in a given culture, (2) resistance to changes in the percentage of positively stained astrocytes after stimulation with either TGF-β1 or TGF-β3, and (3) no positive staining of microglia and oligodendrocytes in cultures isolated from the same region in the CNS (cortex or spinal cord). In doing so, we aimed to investigate protein expression differences between cortical and spinal cord astrocytes and changes in marker expression with TGF-β1 and TGF-β3 stimulation in vitro. We found that only SOX9 in cortical cultures and ALDH1L1 in spinal cord cultures labeled more than 75% of the cells in naïve and stimulated astrocyte cultures and stained less than 5% of the cells in microglia and oligodendrocyte cultures. Further, significantly more cortical than spinal cord astrocytes stained for GFAP, GLAST, ALDH1L1, and SOX9 in naïve cultures, whereas significantly more spinal cord than cortical astrocytes stained for GLAST, GS, and ALDH1L1 in TGF-β1-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. The first sheet in the excel file shows the number of CD68+ microglia and MBP+ oligodendrocytes in each culture condition (naïve or stimulated with TGF-β1 or TGF-β3 in cultures derived from either rat cortex or spinal cord), which was used to calculate the percentage of astrocytes in the cultures. The second sheet in the file shows the number of immunopositive cells counted for each replicate in all culture conditions.