Mass Cytometric Cell Cycle Analysis
The regulated proliferation of cells is a critical factor in tumor progression, antineoplastic therapies, immune system regulation, and the cellular developmental of multicellular organisms. While measurement of cell cycle state by fluorescent flow cytometry is well established, mass cytometry allows the cell cycle to be measured along with large numbers of other antigens enabling characterization of the complex interactions between the cell cycle and 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 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 (pHH3). These measurements can be integrated into a gating strategy that enables clear separation of all five phases of the cell cycle.
Key wordsCell cycle Mass cytometry CyTOF Iodo-deoxyuridine Cyclin Retinoblastoma protein Phosphorylated Histone H3 Ki-67
This chapter has been adapted from a previous chapter .
- 5.Behbehani GK, Samusik N, Bjornson ZB et al (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. https://doi.org/10.1158/2159-8290.cd-15-0298CrossRefPubMedPubMedCentralGoogle Scholar
- 11.Darzynkiewicz Z, Gong J, Juan G et al (1996) Cytometry of cyclin proteins. Cytometry 25(1):1–13. https://doi.org/10.1002/(sici)1097-0320(19960901)25:1<1::aid-cyto1>3.0.co;2-nCrossRefPubMedGoogle Scholar
- 14.Devine RD, Sekhri P, Behbehani GK (2018) Effect of storage time and temperature on cell cycle analysis by mass cytometry. Cytometry 93:1141–1149. https://doi.org/10.1002/cyto.a.23630
- 17.Zunder ER, Finck R, Behbehani GK et al (2015) Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm. Nat Protoc 10(2):316–333. https://doi.org/10.1038/nprot.2015.020. http://www.nature.com/nprot/journal/v10/n2/abs/nprot.2015.020.html#supplementary-informationCrossRefPubMedPubMedCentralGoogle Scholar
- 21.Qiu P, Simonds EF, Bendall SC, et al (2011) Extracting a cellular hierarchy from high-dimensional cytometry data with SPADE. Nat Biotechnol 29(10):886–891. http://www.nature.com/nbt/journal/v29/n10/abs/nbt.1991.html#supplementary-information
- 22.Amir el AD, Davis KL, Tadmor MD et al (2013) viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat Biotechnol 31(6):545–552. https://doi.org/10.1038/nbt.2594