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Cell Cycle Analysis by Mass Cytometry

Part of the Methods in Molecular Biology book series (MIMB,volume 1686)


The regulated progression of cells through the cell cycle during proliferation is a critical factor in tumor progression, anti-neoplastic therapy response, immune system regulation, and developmental biology. While flow cytometric measurement of cell cycle progression is well established, mass cytometry assays allow the cell cycle to be measured along with up to 39 other antigens enabling characterization of the complex interactions between the cell cycle and a wide variety of cellular processes. This method describes the use of mass cytometry for the analysis of cell cycle state for cells from three different sources: in vitro cultured cell lines, ex vivo human blood or bone marrow, and in vivo labeling and ex vivo analysis of murine tissues. The method utilizes incorporation of 5-Iodo-2′-deoxyuridine (IdU), combined with measurement of phosphorylated retinoblastoma protein (pRb), cyclin B1, and phosphorylated histone H3 (p-HH3). These measurements can be integrated into a gating strategy that allows for clear separation of all five phases of the cell cycle.

Key words

  • Cell cycle
  • Mass cytometry
  • CyTOF
  • Iodo-deoxyuridine
  • Cyclin
  • Retinoblastoma protein
  • Phosphorylated histone H3
  • Ki-67

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  1. Darzynkiewicz Z, Juan G, Srour EF (2004) Differential staining of DNA and RNA. Curr Protoc Cytom Chapter 7(Unit 7):3. doi:10.1002/0471142956.cy0703s30

    PubMed  Google Scholar 

  2. Pozarowski P, Darzynkiewicz Z (2004) Analysis of cell cycle by flow cytometry. Methods Mol Biol 281:301–311. doi:10.1385/1-59259-811-0:301

    CAS  PubMed  Google Scholar 

  3. Tanner SD, Baranov VI, Ornatsky OI, Bandura DR, George TC (2013) An introduction to mass cytometry: fundamentals and applications. Cancer Immun, Immunother 62(5):955–965. doi:10.1007/s00262-013-1416-8

    CrossRef  Google Scholar 

  4. Ornatsky OI, Lou X, Nitz M, Schafer S, Sheldrick WS, Baranov VI, Bandura DR, Tanner SD (2008) Study of cell antigens and intracellular DNA by identification of element-containing labels and metallointercalators using inductively coupled plasma mass spectrometry. Anal Chem 80(7):2539–2547. doi:10.1021/ac702128m

    CrossRef  CAS  PubMed  Google Scholar 

  5. Behbehani GK, Bendall SC, Clutter MR, Fantl WJ, Nolan GP (2012) Single-cell mass cytometry adapted to measurements of the cell cycle. Cytometry A 81(7):552–566. doi:10.1002/cyto.a.22075

    CrossRef  PubMed  PubMed Central  Google Scholar 

  6. Raval A, Behbehani GK, Nguyen le XT, Thomas D, Kusler B, Garbuzov A, Ramunas J, Holbrook C, Park CY, Blau H, Nolan GP, Artandi SE, Mitchell BS (2015) Reversibility of defective Hematopoiesis caused by telomere shortening in telomerase knockout mice. PLoS One 10(7):e0131722. doi:10.1371/journal.pone.0131722

    CrossRef  PubMed  PubMed Central  Google Scholar 

  7. Behbehani GK, Samusik N, Bjornson ZB, Fantl WJ, Medeiros BC, Nolan GP (2015) Mass cytometric functional profiling of acute myeloid Leukemia defines cell-cycle and Immunophenotypic properties that correlate with known responses to therapy. Cancer Discov 5(9):988–1003. doi:10.1158/

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  8. Strauss-Albee DM, Fukuyama J, Liang EC, Yao Y, Jarrell JA, Drake AL, Kinuthia J, Montgomery RR, John-Stewart G, Holmes S, Blish CA (2015) Human NK cell repertoire diversity reflects immune experience and correlates with viral susceptibility. Sci Transl Med 7(297):297ra115. doi:10.1126/scitranslmed.aac5722

    CrossRef  PubMed  PubMed Central  Google Scholar 

  9. Chang Q, Ornatsky OI, Koch CJ, Chaudary N, Marie-Egyptienne DT, Hill RP, Tanner SD, Hedley DW (2015) Single-cell measurement of the uptake, intratumoral distribution and cell cycle effects of cisplatin using mass cytometry. Int J Cancer 136(5):1202–1209. doi:10.1002/ijc.29074

    CrossRef  CAS  PubMed  Google Scholar 

  10. Krutzik PO, Nolan GP (2003) Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events. Cytometry A 55(2):61–70. doi:10.1002/cyto.a.10072

    CrossRef  PubMed  Google Scholar 

  11. Darzynkiewicz Z, Gong J, Juan G, Ardelt B, Traganos F (1996) Cytometry of cyclin proteins. Cytometry 25(1):1–13. doi:10.1002/(sici)1097-0320(19960901)25:1<1::aid-cyto1>;2-n

    CrossRef  CAS  PubMed  Google Scholar 

  12. Fienberg HG, Simonds EF, Fantl WJ, Nolan GP, Bodenmiller B (2012) A platinum-based covalent viability reagent for single-cell mass cytometry. Cytometry A 81(6):467–475. doi:10.1002/cyto.a.22067

    CrossRef  PubMed  Google Scholar 

  13. Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1):4–17. doi:10.1002/cyto.a.20167

    CrossRef  PubMed  Google Scholar 

  14. Behbehani GK, Thom C, Zunder ER, Finck R, Gaudilliere B, Fragiadakis GK, Fantl WJ, Nolan GP (2014) Transient partial permeabilization with saponin enables cellular barcoding prior to surface marker staining. Cytometry A 85(12):1011–1019. doi:10.1002/cyto.a.22573

    CrossRef  PubMed  PubMed Central  Google Scholar 

  15. Zunder ER, Finck R, Behbehani GK, Amir E-aD, Krishnaswamy S, Gonzalez VD, Lorang CG, Bjornson Z, Spitzer MH, Bodenmiller B, Fantl WJ, Pe'er D, Nolan GP (2015) Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm. Nat Prot 10(2):316–333. doi:10.1038/nprot.2015.020.

    CrossRef  CAS  Google Scholar 

  16. Ren S, Rollins BJ (2004) Cyclin C/Cdk3 promotes Rb-dependent G0 exit. Cell 117(2):239–251. doi:10.1016/s0092-8674(04)00300-9

    CrossRef  CAS  PubMed  Google Scholar 

  17. Goto H, Yasui Y, Nigg EA, Inagaki M (2002) Aurora-B phosphorylates histone H3 at serine28 with regard to the mitotic chromosome condensation. Genes Cells 7(1):11–17. doi:10.1046/j.1356-9597.2001.00498.x

    CrossRef  CAS  PubMed  Google Scholar 

  18. Hirata A, Inada K, Tsukamoto T, Sakai H, Mizoshita T, Yanai T, Masegi T, Goto H, Inagaki M, Tatematsu M (2004) Characterization of a monoclonal antibody, HTA28, recognizing a histone H3 phosphorylation site as a useful marker of M-phase cells. J Histochem Cytochem 52(11):1503–1509. doi:10.1369/jhc.4A6285.2004

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  19. Qiu P, Simonds EF, Bendall SC, Gibbs KD, Bruggner RV, Linderman MD, Sachs K, Nolan GP, Plevritis SK (2011) Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE. Nat Biotechnol 29(10):886–891.

    CrossRef  CAS  PubMed  PubMed Central  Google Scholar 

  20. Amir el AD, Davis KL, Tadmor MD, Simonds EF, Levine JH, Bendall SC, Shenfeld DK, Krishnaswamy S, Nolan GP, Pe'er D (2013) viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol 31(6):545–552. doi:10.1038/nbt.2594

    CrossRef  Google Scholar 

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Correspondence to Gregory K. Behbehani .

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Behbehani, G.K. (2018). Cell Cycle Analysis by Mass Cytometry. In: Lacorazza, H. (eds) Cellular Quiescence. Methods in Molecular Biology, vol 1686. Humana Press, New York, NY.

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7370-5

  • Online ISBN: 978-1-4939-7371-2

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