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We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.
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.
The molecular complexity of human astrocytes remains poorly defined in primary tissue, requiring better tools for their isolation and characterization at high resolution, both in health and disease. Separation of intact human neurons and glia from their niche has proven difficult due to limited access of fresh brain tissue samples, the heavily interconnected nature of glial and neuronal processes, and inevitable cellular activation during processing, all of which limit the molecular characterization of these cell types ex vivo1. Fluorescence-activated nuclei sorting (FANS) has emerged as an alternative to live-cell sorting, enabling th....
NOTE: The Program for the Protection of Human Subjects at the Icahn School of Medicine at Mount Sinai (ISMMS) and its Institutional Review Board (IRB) assures the ethical conduct of research and compliance with federal, state, and institutional regulations. In this study, all postmortem specimens used were de-identified, obtained under appropriate consent through the biorepository, and were exempt from "human research" designation by ISMMS's IRB (HS#14-01007).
1. Buffer prepara.......
Nuclei were collected from fresh (unfixed) snap-frozen temporal neocortex tissue with a postmortem collection time of 12 h. After tissue dissociation into nuclei suspension, samples were incubated with antibodies against NeuN, PAX6, and OLIG2, and sorted according to the gating shown in Figure 1 and Figure 2. Nuclei were collected from NeuN+, PAX6+NeuN-, and OLIG2+NeuN- sorted populations (Figure 1E,F and
Experimental design following the outlined protocol should be finalized after considering several biological and technical factors. Starting tissue samples are fresh-frozen, without having been fixed, and preferably have a short postmortem collection interval to maximize nuclei recovery. Based on experience, a PMI of up to 24 h allows for adequate nuclei recovery; however, a PMI of 12 h or less is preferable to optimize intact nuclei recovery. Additional factors apart from PMI, including temperature of body storage and p.......
We like to thank members in Pathology and Neurosurgery at the Icahn School of Medicine at Mount Sinai for help with the procurement of de-identified brain tissue and ISMMS's Flow Cytometry CORE for expert advice. The study was partially funded by NIH RF1DA048810, R01NS106229, R03NS101581 (to N.M.T.), and R61DA048207 (to S.A.).
....Name | Company | Catalog Number | Comments |
10x PBS pH 7.2 | Invitrogen | 70013073 | |
ANTI-NEUN ANTIBODY CLONE A60 | Millipore | MAB377A5MI | mouse anti-NeuN conjugated to a fluorescent compound AF555 (excitation, 553 nm; emission, 568 nm) |
ANTI-OLIG2 ANTIBODY CLONE 211 | Millipore | MABN50A4MI | mouse anti-OLIG2 conjugated to a fluorescent compound AF488 (excitation, 499 nm; emission, 520 nm) |
Bovine Serum Albumin | Fisher | BP9704-100 | |
Bright-Line Counting Chamber | Hausser Scientific | 3110V | |
Calcium Chloride Anhydrous | Fisher | C614-3 | |
Cell Strainers, 40 µM | SP Scienceware | 136800040 | |
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Invitrogen | D1306 | |
DL-Dithiothreitol | Sigma | 43815-1G | |
DNA Library Kit | Illumina, Nextera | FC-121–1030 | |
DNAse I | Worthington | LS002139 | |
Dounce Tissue Grinder | WHEATON | 357542 | |
FACS Sorter | BD Biosciences | BD FACSAria III | |
Magnesium Acetate Tetrahydrate | Fisher | M13-500 | |
PAX6 (PAX6/496) - 100 TESTS | Novus | NBP234705J | |
RNA Clean & Concentrator | Zymo Research | R1013 | |
RNaseZap RNase Decontamination Solution | Invitrogen | AM9780 | |
SMARTer Stranded Total RNA-Seq Kit Pico Input Mammalian | Clontech Laboratories | 635005 | Fragmentation time of 2.5 minutes, as recommended for low RIN RNA values. |
Sucrose, crystal certified, ACS, 500 mg | Fisher | S5500 | |
SW 41 Ti Swinging-Bucket Rotor | Beckman Coulter | 331362 | |
Tris-HCl, 1M Solution, pH 8.0, Molecular Biology Grade, Ultrapure | Thermo Scientific | J22638AE | |
TritonX-100 | Fisher | BP151-500 | non-ionic surfactant in lysis buffer |
TRIzol LS Reagent | Invitrogen | 10296028 | |
TRIzol Reagent | Invitrogen | 15596026 | reagent for isolation of RNA |
Trypan Blue Solution, 0.4% | Gibco | 15250061 | |
Ultracentrifuge | Beckman Coulter Optima XE-100 | A94516 | |
Ultracentrifuge tubes PP 9/16 X 3-1/2 | Beckman Coulter | 331372 | |
UltraPure Distilled Water (RNAse-, DNAse-free) | Invitrogen | 10977023 | referred to as distilled water |
Ultrapure EDTA | Life Technologies | 15576-028 |
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