Figure 1: Validation of neuron- and astrocyte-enriched isolation by FANS.

(A–F) Representative examples of sequential FANS gating to exclude debris and doublets (A–C) and define background staining using (D) a DAPI-only control for the collection of (E–F) enriched neuronal (NeuN+), astrocyte (PAX6+NeuN−), and OPC (OLIG2++NeuN−) nuclei populations. (G) Minimal qPCR panel using pan-astrocyte (GFAP, ALDH1L1), neuronal (RBFOX3), and OPC (PDGFRA) markers for quality control of the collected populations (A–G: temporal neocortex autopsy specimen without pathology, 12 h PMI, 100,000 events shown; dotted lines represent positively gated sequential populations; solid lines represent final collection of cell-type enriched populations). (H) Heatmap generated from row-normalized bulk RNA sequencing data showing expression of canonical astrocyte and neuronal markers in PAX6+ and NeuN+ sorted populations (n=3 autopsy cases, temporal neocortex without pathology, PMI 12–21 h). (I) Representative immunofluorescence image of PAX6, GFAP, and NeuN in human neocortex. Arrows indicate astrocytes co-expressing PAX6 and GFAP. Abbreviations: FANS = fluorescence-activate nuclei sorting; DAPI = 4′,6-diamidino-2-phenylindole; NeuN = neuronal nuclei; PAX6 = paired box protein 6; OLIG2 = oligodendrocyte transcription factor 2; OPC = oligodendrocyte progenitor cell; qPCR = quantitative polymerase chain reaction; GFAP = glial fibrillary acidic protein; ALDH1L1 = 10-formyltetrahydrofolate dehydrogenase; RBFOX3 = RNA-binding protein FOX-1 homolog 3; PDGFRA = platelet-derived growth factor alpha; PMI = postmortem interval; SSC-A = side scatter-area; FSC-A = forward scatter area; SSC-W = side scatter width; FSC-W = forward scatter width; NeuN-555 = mouse anti-NeuN conjugated to AF555; PAX6-APC = mouse anti-PAX6 conjugated to allophycocyanin; OLIG2-488 = mouse anti-OLIG2 conjugated to green fluorescent dye for the 488 nm laser line.