ArticleJournal of visualized experiments : JoVE2021
Isolation of Adult Human Astrocyte Populations from Fresh-frozen Cortex using Fluorescence-Activated Nuclei Sorting.
Article in Journal of visualized experiments : JoVE, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Who cites it
6 citing papers in PubMed.
- Writing the Engram: Epigenetic Mechanisms of Memory Allocation.Journal of neurochemistry · 2025Review
- Review
- Fluorescence-activated nuclear sorting (FANS) of nuclei from in vitro-generated syncytiotrophoblast.Placenta · 2025Article
- Article
- An atlas of late prenatal human neurodevelopment resolved by single-nucleus transcriptomics.Nature communications · 2022Article
- Ghrelin Regulates Expression of the Transcription Factor Pax6 in Hypoxic Brain Progenitor Cells and Neurons.Cells · 2022Article
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5 authors.
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Abstract
The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen postmortem human temporal neocortex tissue. This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.
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