Flow Cytometric and Sorting Analyses for Nuclear DNA Content, Nucleotide Sequencing, and Interphase FISH

  • Gwendolyn E. Kaeser
  • Jerold ChunEmail author
Part of the Neuromethods book series (NM, volume 131)


The study of genomic mosaicism among human brain cells is challenging. The human brain contains hundreds of billions of cells that are intricately connected and difficult to separate as intact, single cells. Additional challenges are encountered when interrogating small, seemingly random changes within single-cell genomes. Flow cytometric analysis (FCM), and fluorescence-activated nuclear sorting (FANS), has expanded our assessment capabilities for global and specific genomic and transcriptomic changes in human brain cells. The general approach is being utilized in a variety of downstream applications by many laboratories. Here we provide detailed methods of nuclear DNA content assessment and sorting that reports population averages as well as single-cell nuclear DNA content from cells of the human brain. We highlight protocol modifications that allow the same nuclear preparation to be used for subpopulation-specific FANS (also see chapter “Single-Cell Whole Genome Amplification and Sequencing to Study Neuronal Mosaicism and Diversity”) in downstream analyses such as fluorescent in situ hybridization (FISH) (see chapters “FISH-Based Assays for Detecting Genomic (Chromosomal) Mosaicism in Human Brain Cells,” “FISH Analysis of Aging-Associated Aneuploidy in Neurons and Non-neuronal Brain Cells” and “Using Fluorescence In Situ Hybridization (FISH) Analysis to Measure Chromosome Instability and Mosaic Aneuploidy in Neurodegenerative Diseases”), and single-cell genomic and transcriptomic sequencing (see chapters “Flow Cytometric Quantification, Isolation, and Subsequent Epigenetic Analysis of Tetraploid Neurons,” “Single Cell CNV Detection in Human Neuronal Nuclei,” “Multiple Annealing and Looping-Based Amplification Cycles (MALBAC) for the Analysis of DNA Copy Number Variation,” and “Single-Cell Whole Genome Amplification and Sequencing to Study Neuronal Mosaicism and Diversity”). Other downstream techniques include, but are not limited to, single-cell qPCR (see chapter “Competitive PCR for Copy Number Assessment by Restricting dNTPs”) and estimation of line-1 copy number (see chapters “Analysis of LINE-1 Retrotransposition in Neural Progenitor Cells and Neurons,” “Estimation of LINE-1 Copy Number in the Brain Tissue and Isolated Neuronal Nuclei,” and “Analysis of Somatic LINE-1 Insertions in Neurons”).

Key words

DNA content variation Flow sorting Flow cytometry Neuron Nuclei NeuN Sequencing Fish Genomic mosaicism Somatic Aneuploidy Aneusomy 



The authors are funded by the NIH Common Fund Single Cell Analysis Program (1U01MH098977), the NIAAA (4R01AA021402), and the Neuroplasticity of Aging Training Grant (5T32AG000216). We thank Danielle Jones, Dr. Ming Hsiang Lee, and Suzanne Rohrback for their contributions to this manuscript.


  1. 1.
    Rehen SK, McConnell MJ, Kaushal D, Kingsbury MA, Yang AH, Chun J (2001) Chromosomal variation in neurons of the developing and adult mammalian nervous system. Proc Natl Acad Sci U S A 98(23):13361–13366. doi: 10.1073/pnas.231487398 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kaushal D, Contos JJ, Treuner K, Yang AH, Kingsbury MA, Rehen SK, McConnell MJ, Okabe M, Barlow C, Chun J (2003) Alteration of gene expression by chromosome loss in the postnatal mouse brain. J Neurosci 23(13):5599–5606PubMedGoogle Scholar
  3. 3.
    Yang AH, Kaushal D, Rehen SK, Kriedt K, Kingsbury MA, McConnell MJ, Chun J (2003) Chromosome segregation defects contribute to aneuploidy in normal neural progenitor cells. J Neurosci 23(32):10454–10462PubMedGoogle Scholar
  4. 4.
    Kingsbury MA, Friedman B, McConnell MJ, Rehen SK, Yang AH, Kaushal D, Chun J (2005) Aneuploid neurons are functionally active and integrated into brain circuitry. Proc Natl Acad Sci U S A 102(17):6143–6147. doi: 10.1073/pnas.0408171102 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Rehen SK, Yung YC, McCreight MP, Kaushal D, Yang AH, Almeida BS, Kingsbury MA, Cabral KM, McConnell MJ, Anliker B, Fontanoz M, Chun J (2005) Constitutional aneuploidy in the normal human brain. J Neurosci 25(9):2176–2180. doi: 10.1523/JNEUROSCI.4560-04.2005 CrossRefPubMedGoogle Scholar
  6. 6.
    Yurov YB, Iourov IY, Monakhov VV, Soloviev IV, Vostrikov VM, Vorsanova SG (2005) The variation of aneuploidy frequency in the developing and adult human brain revealed by an interphase FISH study. J Histochem Cytochem 53(3):385–390. doi: 10.1369/jhc.4A6430.2005 CrossRefPubMedGoogle Scholar
  7. 7.
    Yurov YB, Iourov IY, Vorsanova SG, Liehr T, Kolotii AD, Kutsev SI, Pellestor F, Beresheva AK, Demidova IA, Kravets VS, Monakhov VV, Soloviev IV (2007) Aneuploidy and confined chromosomal mosaicism in the developing human brain. PLoS One 2(6):e558. doi: 10.1371/journal.pone.0000558 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Peterson SE, Westra JW, Paczkowski CM, Chun J (2008) Chromosomal mosaicism in neural stem cells. Methods Mol Biol 438:197–204. doi: 10.1007/978-1-59745-133-8_16 CrossRefPubMedGoogle Scholar
  9. 9.
    Westra JW, Peterson SE, Yung YC, Mutoh T, Barral S, Chun J (2008) Aneuploid mosaicism in the developing and adult cerebellar cortex. J Comp Neurol 507(6):1944–1951. doi: 10.1002/cne.21648 CrossRefPubMedGoogle Scholar
  10. 10.
    Peterson SE, Yang AH, Bushman DM, Westra JW, Yung YC, Barral S, Mutoh T, Rehen SK, Chun J (2012) Aneuploid cells are differentially susceptible to caspase-mediated death during embryonic cerebral cortical development. J Neurosci 32(46):16213–16222. doi: 10.1523/JNEUROSCI.3706-12.2012 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Westra JW, Rivera RR, Bushman DM, Yung YC, Peterson SE, Barral S, Chun J (2010) Neuronal DNA content variation (DCV) with regional and individual differences in the human brain. J Comp Neurol 518(19):3981–4000. doi: 10.1002/cne.22436 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Gole J, Gore A, Richards A, Chiu YJ, Fung HL, Bushman D, Chiang HI, Chun J, Lo YH, Zhang K (2013) Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nat Biotechnol 31(12):1126–1132. doi: 10.1038/nbt.2720 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    McConnell MJ, Lindberg MR, Brennand KJ, Piper JC, Voet T, Cowing-Zitron C, Shumilina S, Lasken RS, Vermeesch JR, Hall IM, Gage FH (2013) Mosaic copy number variation in human neurons. Science 342(6158):632–637. doi: 10.1126/science.1243472 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Cai X, Evrony GD, Lehmann HS, Elhosary PC, Mehta BK, Poduri A, Walsh CA (2015) Single-cell, genome-wide sequencing identifies clonal somatic copy-number variation in the human brain. Cell Rep 10(4):645. doi: 10.1016/j.celrep.2014.07.043 CrossRefPubMedGoogle Scholar
  15. 15.
    Bushman DM, Kaeser GE, Siddoway B, Westra JW, Rivera RR, Rehen SK, Yung YC, Chun J (2015) Genomic mosaicism with increased amyloid precursor protein (APP) gene copy number in single neurons from sporadic Alzheimer’s disease brains. eLife 4. doi: 10.7554/eLife.05116
  16. 16.
    Lodato MA, Woodworth MB, Lee S, Evrony GD, Mehta BK, Karger A, Lee S, Chittenden TW, D'Gama AM, Cai X, Luquette LJ, Lee E, Park PJ, Walsh CA (2015) Somatic mutation in single human neurons tracks developmental and transcriptional history. Science 350(6256):94–98. doi: 10.1126/science.aab1785 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Hazen JL, Faust GG, Rodriguez AR, Ferguson WC, Shumilina S, Clark RA, Boland MJ, Martin G, Chubukov P, Tsunemoto RK, Torkamani A, Kupriyanov S, Hall IM, Baldwin KK (2016) The complete genome sequences, unique mutational spectra, and developmental potency of adult neurons revealed by cloning. Neuron 89(6):1223–1236. doi: 10.1016/j.neuron.2016.02.004 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Lake BB, Ai R, Kaeser GE, Salathia NS, Yung YC, Liu R, Wildberg A, Gao D, Fung HL, Chen S, Vijayaraghavan R, Wong J, Chen A, Sheng X, Kaper F, Shen R, Ronaghi M, Fan JB, Wang W, Chun J, Zhang K (2016) Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain. Science 352(6293):1586–1590. doi: 10.1126/science.aaf1204 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Krishan A (1975) Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J Cell Biol 66(1):188–193CrossRefPubMedGoogle Scholar
  20. 20.
    Riccardi C, Nicoletti I (2006) Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat Protoc 1(3):1458–1461. doi: 10.1038/nprot.2006.238 CrossRefPubMedGoogle Scholar
  21. 21.
    Dolezel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2(9):2233–2244. doi: 10.1038/nprot.2007.310 CrossRefPubMedGoogle Scholar
  22. 22.
    Darzynkiewicz Z, Huang X (2004) Analysis of cellular DNA content by flow cytometry. Curr Protoc Immunol Chapter 5:unit 5.7. doi: 10.1002/0471142735.im0507s60 PubMedGoogle Scholar
  23. 23.
    Darzynkiewicz Z (2011) Critical aspects in analysis of cellular DNA content. Curr Protoc Cytom Chapter 7:unit 7.2. doi: 10.1002/0471142956.cy0702s56 PubMedGoogle Scholar
  24. 24.
    Mullen RJ, Buck CR, Smith AM (1992) NeuN, a neuronal specific nuclear protein in vertebrates. Development 116(1):201–211PubMedGoogle Scholar
  25. 25.
    Fan J, Salathia N, Liu R, Kaeser GE, Yung YC, Herman JL, Kaper F, Fan JB, Zhang K, Chun J, Kharchenko PV (2016) Characterizing transcriptional heterogeneity through pathway and gene set overdispersion analysis. Nat Methods 13(3):241–244. doi: 10.1038/nmeth.3734 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Evrony GD, Cai X, Lee E, Hills LB, Elhosary PC, Lehmann HS, Parker JJ, Atabay KD, Gilmore EC, Poduri A, Park PJ, Walsh CA (2012) Single-neuron sequencing analysis of L1 retrotransposition and somatic mutation in the human brain. Cell 151(3):483–496. doi: 10.1016/j.cell.2012.09.035 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Evrony GD, Lee E, Mehta BK, Benjamini Y, Johnson RM, Cai X, Yang L, Haseley P, Lehmann HS, Park PJ, Walsh CA (2015) Cell lineage analysis in human brain using endogenous retroelements. Neuron 85(1):49–59. doi: 10.1016/j.neuron.2014.12.028 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Fischer HG, Morawski M, Bruckner MK, Mittag A, Tarnok A, Arendt T (2012) Changes in neuronal DNA content variation in the human brain during aging. Aging Cell 11(4):628–633. doi: 10.1111/j.1474-9726.2012.00826.x CrossRefPubMedGoogle Scholar
  29. 29.
    Arendt T, Bruckner MK, Losche A (2015) Regional mosaic genomic heterogeneity in the elderly and in Alzheimer’s disease as a correlate of neuronal vulnerability. Acta Neuropathol 130(4):501–510. doi: 10.1007/s00401-015-1465-5 CrossRefPubMedGoogle Scholar
  30. 30.
    Vindeløv LL, Christensen IJ, Nissen NI (1983) Standardization of high resolution flow cytometric DNA analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards. Cytometry 3(5):328–331. doi: 10.1002/cyto.990030504 CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  1. 1.Sanford Burnham Prebys Medical Discovery InstituteLa JollaUSA
  2. 2.Biomedical Sciences Graduate ProgramUniversity of California San DiegoSan DiegoUSA

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