CyTOF Mass Cytometry for Click Proliferation Assays

Vinko Tosevski1, Egor Ulashchik2, Andrea Trovato3, Paolo Cappella3

1 Mass Cytometry Facility, University of Zurich, Zurich, 2 Primetech ALC, Minsk, 3 FlowMetric Europe SpA, Lodi, Milan
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.50
DOI:  10.1002/cpcy.25
Online Posting Date:  July, 2017
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Abstract

Novel cell analyzers, including polychromatic flow cytometers and isotopical cytometry by time of flight (CyTOF) mass cytometers, enable simultaneous measurement of virtually bondless characteristics at the single‐cell level. BrdU assays for quantifying cellular proliferation are common but have several limitations, including the need for a DNA denaturation step and inability to simultaneously resolve multiple parameters and phenotypic complexity. Click chemistry reactions have become popular in the past decade, as they can resolve these issues. This protocol introduces a novel assay able to bridge flow cytometry and CyTOF analysis for active S‐phase determination in cell cycle applications, combining well‐established click chemistry with a novel iodo‐deoxyuridine (IdU) azide derivative and a cross‐reactive anti‐IdU antibody for detecting incorporated EdU during DNA synthesis. This method is preferred over traditional BrdU‐based assays for complex and multiparametric experiments. It provides a feasible cost‐effective approach for detecting ethynyl‐labeled nucleotides, with the advantage of combining flow and mass cytometry analyses. © 2017 by John Wiley & Sons, Inc.

Keywords: BrdU; click chemistry; cell cycle; CyTOF; IdU azide; IMA; mass cytometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Click Chemistry Using 5′‐Azido‐IdU for Ethynyl Uridine Detection by Anti‐IdU Antibody and 127I Detection
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Click Chemistry Using 5′‐Azido‐IdU for Ethynyl Uridine Detection by Anti‐IdU Antibody and 127I Detection

  Materials
  • Suspension cell culture (e.g., Jurkat cells) in appropriate culture medium
  • Ethynyl nucleotide (select one):
    • 10 mM EdU (Berry & Associates, PY7562) in DMSO
    • 10 mM EdC (Sigma‐Aldrich, T511307) in DMSO
    • 40 mM F‐ara‐EdU (Sigma‐Aldrich, T511293) in DMSO
  • Control nucleotides:
    • 3 mM BrdU (Sigma‐Aldrich, B5002) in water
    • 25 mM IdU (Berry & Associates, PY7615) in DMSO
    • 25 mM IdC (Berry & Associates, PY7607) in DMSO
  • Phosphate‐buffered saline (PBS) without calcium and magnesium (Thermo Fisher Scientific)
  • 100% methanol (Sigma‐Aldrich)
  • BSA Stain Buffer (BD Biosciences)
  • 0.1% Triton X‐100 (Sigma) in PBS
  • 10 mM 5‐iodo‐5′‐azido‐2′,5′‐dideoxyuridine (IMA; HPLC >97%) in DMSO
  • 10 mM 5‐bromo‐3′‐azido‐2′,3′‐dideoxyuridine (BMA; Berry & Associates, PY7286) in DMSO
  • 0.1 M copper(II) sulfate solution (Fluka, 35185)
  • 0.5 M sodium‐L‐ascorbate (see recipe; also see unit 7.34), freshly prepared
  • SBT: BSA Stain Buffer with 0.5% Tween‐20 (Sigma‐Aldrich), freshly prepared
  • Antibodies:
    • 0.025 mg/ml anti‐BrdU monoclonal mouse antibody (clone B44, BD Biosciences)
    • Alexa Fluor 488–conjugated goat anti‐mouse IgG (H+L, Thermo Fisher Scientific, A‐11001)
  • PI staining solution (see recipe)
  • Cell‐ID Intercalator‐Ir (Fluidigm, 201192 A or 201192B)
  • Cell‐ID Cisplatin (Fluidigm, 201064)
  • 15‐ml screw‐cap centrifuge tubes
  • Vacuum aspirator (e.g., Costar)
  • Hemocytometer
  • Shaker
  • 5‐ml (12 × 75–mm) polystyrene tubes (BD Falcon) for FCM
  • Flow cytometer with excitation at 488 nm (e.g., Beckton Dickinson BD FACSAria III)
  • 2‐ml microcentrifuge tubes
  • MaxPar Cell Staining Buffer (Fluidigm, 201068)
  • MaxPar Fix and Perm Buffer (Fluidigm, 201067)
  • Cell‐ID Cisplatin (Fluidigm, 201064, optional)
  • Cell‐ID Intercalator‐Ir (Fluidigm, 201192 A or 201192B)
  • EQ Four Element Calibration Beads (Fluidigm, 201078)
  • 5‐ml (12 × 75–mm) polystyrene round‐bottom tubes with cell‐strainer caps (BD Falcon, 352235)
  • CyTOF mass cytometer (e.g., CyTOF 2, Fluidigm)
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Figures

Videos

Literature Cited

  Bandura, D. R., Baranov, V. I., Ornatsky, O. I., Antonov, A., Kinach, R., Lou, X., … Tanner, S. D. (2009). Mass cytometry: Technique for real time single cell multitarget immunoassay based on inductively coupled plasma time‐of‐flight mass spectrometry. Analytical Chemistry, 81, 6813–6822. doi: 10.1021/ac901049w.
  Behbehani, G. K., Bendall, S. C., Clutter, M. R., Fantl, W. J., & Nolan, G. P. (2012). Single‐cell mass cytometry adapted to measurements of the cell cycle. Cytometry, Part A, 81, 552–566. doi: 10.1002/cyto.a.22075.
  Bodenmiller, B., Zunder, E. R., Finck, R., Chen, T. J., Savig, E. S., Bruggner, R. V., … Nolan, G. P. (2012). Multiplexed mass cytometry profiling of cellular states perturbed by small‐molecule regulators. Nature Biotechnology, 30, 858–867. doi: 10.1038/nbt.2317.
  Cappella, P., Gasparri, F., Pulici, M., & Moll, J. (2015a). Cell proliferation method: Click chemistry based on BrdU coupling for multiplex antibody staining. Current Protocols in Cytometry, 72, 7.34.1‐7.34.17. doi: 10.1002/0471142956.cy0734s72.
  Cappella, P., Gasparri, F., Pulici, M., & Moll, J. (2008). A novel method based on click chemistry, which overcomes limitations of cell cycle analysis by classical determination of BrdU incorporation, allowing multiplex antibody staining. Cytometry, Part A, 73, 626–636. doi: 10.1002/cyto.a.20582.
  Cappella, P., Giansanti, V., Pulici, M., & Gasparri, F. (2013). From “click” to “Fenton” chemistry for 5‐bromo‐2′‐deoxyuridine determination. Cytometry, Part A, 83, 989–1000. doi: 10.1002/cyto.a.22343.
  Cappella, P., Pulici, M., & Gasparri, F. (2015b). Application of click chemistry conditions for 5‐bromo‐2′‐deoxyuridine determination through Fenton and related reactions. Current Protocols in Cytometry, 71, 7.43.1–7.43.17. doi: 10.1002/0471142956.cy0743s71.
  Cappella, P., Tomasoni, D., Faretta, M., Lupi, M., Montalenti, F., Viale, F., … Ubezio, P. (2001). Cell cycle effects of gemcitabine. International Journal of Cancer, 93, 401–408. doi: 10.1002/ijc.1351.
  Cheng, B., Xie, R., Dong, L., & Chen, X. (2016). Metabolic remodeling of cell‐surface sialic acids: Principles, applications, and recent advances. ChemBioChem, 17, 11–27. doi: 10.1002/cbic.201500344.
  Darzynkiewicz, J., & Juan, G. (2001). Analysis of DNA content and BrdU incorporation. Current Protocols in Cytometry, 2, 7.7.1–7.7.9. doi: 10.1002/0471142956.cy0707s02.
  Di Palma, S., & Bodenmiller, B. (2015). Unraveling cell populations in tumors by single‐cell mass cytometry. Current Opinion in Biotechnology, 31, 122–129. doi: 10.1016/j.copbio.2014.07.004.
  Gratzner, H. G. (1982). Monoclonal antibody to 5‐bromo‐ and 5‐iododeoxyuridine: A new reagent for detection of DNA replication. Science, 218(4571), 474–475. doi: 10.1126/science.7123245.
  Guan, L., van der Heijden, G. W., Bortvin, A., & Greenberg, M. M. (2011). Intracellular detection of cytosine incorporation in genomic DNA by using 5‐ethynyl‐2′‐deoxycytidine. Chembiochem, 12, 2184–2190. doi: 10.1002/cbic.201100353.
  Holstein, J. M., & Rentmeister, A. (2016). Current covalent modification methods for detecting RNA in fixed and living cells. Methods, 98, 18–25. doi: 10.1016/j.ymeth.2015.11.016.
  Hou, X., Chai, C., Qian, Q., Li, C., & Chen, Q. (1997). Determination of bromine and iodine in normal tissues from Beijing healthy adults. Biological Trace Element Research, 56, 225–230. doi: 10.1007/BF02785395.
  Hou, X., Hansen, V., Aldahan, A., Possnert, G. R., Lind, O. C., & Lujaniene, G. (2009). A review on speciation of iodine‐129 in the environmental and biological samples. Analytica Chimica Acta, 632, 181–196. doi: 10.1016/j.aca.2008.11.013.
  Leif, R. C., Stein, J. H., & Zucker, R. M. (2004). A short history of the initial application of anti‐5‐BrdU to the detection and measurement of S phase. Cytometry, Part A, 58, 45–52. doi: 10.1002/cyto.a.20012.
  Leipold, M. D., Newell, E. W., & Maecker, H. T. (2015). Multiparameter phenotyping of human PBMCs using mass cytometry. Methods in Molecular Biology, 1343, 81–95. doi: 10.1007/978‐1‐4939‐2963‐4_7.
  Ligasova, A., Liboska, R., Friedecky, D., Micova, K., Adam, T., Ozdian, T., … Koberna, K. (2016). Dr Jekyll and Mr Hyde: A strange case of 5‐ethynyl‐2′‐deoxyuridine and 5‐ethynyl‐2′‐deoxycytidine. Open Biology, 6, 150172. doi: 10.1098/rsob.150172.
  Ligasova, A., Liboska, R., Rosenberg, I., & Koberna, K. (2015). The fingerprint of anti‐bromodeoxyuridine antibodies and its use for the assessment of their affinity to 5‐bromo‐2′‐deoxyuridine in cellular DNA under various conditions. PLoS One, 10, e0132393. doi: 10.1371/journal.pone.0132393.
  Lin, T. S., Neenan, J. P., Cheng, Y. C., & Prusoff, W. H. (1976). Synthesis and antiviral activity of 5‐ and 5′‐substituted thymidine analogs. Journal of Medicinal Chemistry, 19, 495–498. doi: 10.1021/jm00226a009.
  Liu, Z., & Sadler, P. J. (2014). Organoiridium complexes: Anticancer agents and catalysts. Accounts of Chemical Research, 47, 1174–1185. doi: 10.1021/ar400266c.
  Miller, D. D., & Rutzke, M. A. (2010). Atomic absorption spectroscopy, atomic emission spectroscopy, and inductively coupled plasma‐mass spectrometry. In S. S. Nielsen (Ed.), Food Analysis (pp. 421‐442). Springer, U.S. doi: 10.1007/978‐1‐4419‐1478‐1_24.
  Mueller, L., Traub, H., Jakubowski, N., Drescher, D., Baranov, V. I., & Kneipp, J. (2014). Trends in single‐cell analysis by use of ICP‐MS. Analytical and Bioanalytical Chemistry, 406, 6963–6977. doi: 10.1007/s00216‐014‐8143‐7.
  Neef, A. B., & Luedtke, N. W. (2011). Dynamic metabolic labeling of DNA in vivo with arabinosyl nucleosides. Proceedings of the National Academy of Sciences of the United States of America, 108, 20404–20409. doi: 10.1073/pnas.1101126108.
  Nikić, I., & Lemke, E. A. (2015). Genetic code expansion enabled site‐specific dual‐color protein labeling: Superresolution microscopy and beyond. Current Opinion in Chemical Biology, 28, 164–173. doi: 10.1016/j.cbpa.2015.07.021.
  Profrock, D., & Prange, A. (2013). Inductively coupled plasma mass spectrometry (ICP‐MS) for quantitative analysis in environmental and life sciences: A review of challenges, solutions, and trends. Applied Spectroscopy, 66, 843–868. doi: 10.1366/12‐06681.
  Qu, D., Wang, G., Wang, Z., Zhou, L., Chi, W., Cong, S., … Zhang, B. (2011). 5‐Ethynyl‐2′‐deoxycytidine as a new agent for DNA labeling: Detection of proliferating cells. Analytical Biochemistry, 417, 112–121. doi: 10.1016/j.ab.2011.05.037.
  Schafen, S., & Sheldrick, W. S. (2007). Coligand tuning of the DNA binding properties of half‐sandwich organometallic intercalators: Influence of polypyridyl (pp) and monodentate ligands (L = Cl,(NH2)2CS,(NMe2)2CS) on the intercalation of (H5‐pentamethylcyclopentadienyl)‐iridium(III)‐dipyridoquinoxaline and ‐dipyridophenazine complexes. Journal of Organometallic Chemistry, 692, 1300–1309. doi: 10.1016/j.jorganchem.2006.10.033.
  Stern, A. D., Rahman, A. H., & Birtwistle, M. R. (2017). Cell size assays for mass cytometry. Cytometry A, 91, 14–24. doi: 10.1002/cyto.a.23000.
  Sumatoh, H. R., Teng, K. W., Cheng, Y., & Newell, E. W. (2016). Optimization of mass cytometry sample cryopreservation after staining. Cytometry, Part A, 91, 48–61. doi: 10.1002/cyto.a.23014.
  Tavarini, S., Sammicheli, C., Rosa, D., Bertholet, S., & Nuti, S. (2015). New application of CyTOF technology: Quantitative analysis of T cell antigen specific cell proliferation by Click‐iT EdU. Cytoconference (CYTO) 2015, poster B279–410. Glasgow Scotland: International Society for the Advancement of Cytometry.
Internet Resources
  https://www.fluidigm.com/products/helios
  Fluidigm website.
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