A One‐Step Imaging Assay to Monitor Cell Cycle State and Apoptosis in Mammalian Cells

Yangzhong Tang1

1 Sanofi, Cambridge, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130140
Online Posting Date:  March, 2014
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High‐content screening (HCS; fluorescence microscopy with multiple markers followed by automated image analysis) is gaining popularity in drug discovery due to the rich information it reveals about drug responses. It is particularly useful in studying anti‐mitotic drug responses since mitotic arrest provides an activity biomarker. One conventional way to probe mitotic arrest and downstream apoptosis response is to use mitosis‐ and apoptosis‐specific antibodies in cell‐based imaging assays. However, weakly attached cells, especially dead cells, are mostly washed out during antibody labeling steps. Here, we report a rapid and convenient one‐step cell‐imaging assay that accurately measures cell‐cycle state and apoptosis in mammalian cells. The assay uses three fluorescent dyes to stain living cells, involves no wash, and is fixable after live‐cell labeling. Compared to the antibody‐based method, this assay is quicker, more cost‐effective, and yields more accurate dose‐response results. Curr. Protoc. Chem. Biol. 6:1‐5. © 2014 by John Wiley & Sons, Inc.

Keywords: high‐content screening; imaging assay; mitosis; apoptosis; dose response; pharmacology

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Table of Contents

  • Commentary
  • Literature Cited
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Basic Protocol 1:

  • Human cancer cell lines
  • 0.5% Trypsin‐EDTA
  • Growth medium (appropriate for the cell type used)
  • Small‐molecule compounds to be screened, diluted in dimethyl sulfoxide (DMSO)
  • 4× cocktail of cell‐staining reagents, prepared in phosphate‐buffered saline (PBS; pH 7.4):
    • 1 µg/ml LysoTracker‐Red (Invitrogen, cat. no. L‐7528)
    • 4 µg/ml Hoechst 33342 (Sigma, cat. no. B2261)
    • 2 µM DEVD‐NucView488 Caspase‐3 substrate (Biotium, cat. no. 10402)
  • 2% formaldehyde solution: dilute 37% formaldehyde solution (Sigma Aldrich, cat. no. F8775) in phosphate‐buffered saline (PBS)
  • 75‐cm2 tissue culture flasks
  • 50‐ml tubes (e.g., Falcon)
  • Clear‐bottom, black 384‐well imaging plates (Corning, cat. no. 3712)
  • Matrix WellMate (for liquid dispensing; Rudnicki and Johnston, )
  • Cell culture incubator (37°C, 5% CO 2)
  • Epson Compound Transfer Robot (for compound transfer; Rudnicki and Johnston, )
  • Benchtop centrifuge
  • Aluminum plate seals (Corning, cat. no. 6570)
  • Molecular Devices ImageXpress Micro microscope (for multi‐well microplate imaging)
  • Analysis tool written in MATLAB, available through GitHub (https://github.com/xietiao/Tang_et_al_LINCS_cell_scoring; see Tang et al., )
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Literature Cited

Literature Cited
  Bullen, A. 2008. Microscopic imaging techniques for drug discovery. Nat. Rev. Drug Discov. 7:54‐67.
  Lang, P., Yeow, K., Nichols, A., and Scheer, A. 2006. Cellular imaging in drug discovery. Nat. Rev. Drug Discov. 5:343‐356.
  Letai, A.G. 2008. Diagnosing and exploiting cancer's addiction to blocks in apoptosis. Nat. Rev. Cancer 8:121‐132.
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  Madian, A.G., Wheeler, H.E., Jones, R.B., and Dolan, M.E. 2012. Relating human genetic variation to variation in drug responses. Trends Genet. 28:487‐495.
  Rudnicki, S. and Johnston, S. 2009. Overview of liquid handling instrumentation for high‐throughput screening applications. Curr. Protoc. Chem. Biol. 1:43‐54.
  Smyth, M.J., Krasovskis, E., Sutton, V.R., and Johnstone, R.W. 1998. The drug efflux protein, P‐glycoprotein, additionally protects drug‐resistant tumor cells from multiple forms of caspase‐dependent apoptosis. Proc. Natl. Acad. Sci. U.S.A. 95:7024‐7029.
  Tang, Y., Xie, T., Florian, S., Moerke, N., Shamu, C., Benes, C., and Mitchison, T.J. 2013. Differential determinants of cancer cell insensitivity to antimitotic drugs discriminated by a one‐step cell imaging assay. J. Biomol. Screen. 18:1062‐1071.
  Zanella, F., Lorens, J.B., and Link, W. 2010. High content screening: Seeing is believing. Trends Biotechnol. 28:237‐245.
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