Abstract—Objective: This study presents a method for separating NeuN-stained neurons and glial cells in the human fetal cerebral cortex using flow cytometry. NeuN neuronal nuclear antigen is widely used as a universal neuron-specific marker. Antibodies against this protein allows to specifically identify neurons, since this protein is not found in glial cells. The application of this marker allows precise determination of the cellular composition in different brain regions. Methods: This study uses protocols NeuN-based immunohistochemical neuronal quantification for flow cytometry to distinguish neurons and glial cells in different regions of human fetal cerebral cortex. Results: Immunohistochemical staining was optimized to establish a validated antibody concentration for subsequent flow cytometry. Then two parahippocampal cortical sample suspensions stained with DAPI and NeuN were analyzed. We acquired three subpopulations: DAPI–/AF488+, DAPI–/AF488–, and DAPI+/AF488– with variable intensities. Fluorescence microscopy validation detected nuclei matching all cytometry subpopulations. Conclusion: Flow cytometric approach offers single-nucleus resolution for detecting rare populations. Along with
immunohistochemical staining, flow cytometry enhances quantitative and qualitative analysis of neural populations and allows to calculate glia-neuron index which is a critical metric for understanding brain organization, development, and plasticity.