Zinc Fixation for Flow Cytometry Analysis of Intracellular and Surface Epitopes, DNA Content, and Cell Proliferation

Rikke Christensen1, David M. Owens2, Anette Thomsen3, Søren Pedersen1, Uffe Birk Jensen3

1 Department of Clinical Genetics, Aarhus University Hospital, Aarhus, Denmark, 2 Departments of Dermatology and Pathology, College of Physicians and Surgeons, Columbia University, New York, New York, 3 Department of Human Genetics, Aarhus University, Aarhus, Denmark
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 7.40
DOI:  10.1002/0471142956.cy0740s57
Online Posting Date:  July, 2011
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Zinc salt‐based fixation (ZBF) is a simple, cost‐effective, and nonhazardous fixation method for cell suspensions that preserves all cellular structures and enables flow cytometric analysis of both surface and intracellular proteins, DNA content profiles, and pulse‐labeling using the thymidine analog EdU in the same cell sample. ZBF performs equally well to formaldehyde in the preservation of surface epitope labeling and forward and side light scatter parameters, as measured by flow cytometry. DNA is maintained at high molecular weight, improving the quantification and allowing subsequent quantitative PCR analysis. Finally, ZBF treatment allows for long‐term storage of labeled cells with little change in these parameters. Curr. Protoc. Cytom. 57:7.40.1‐7.40.9. © 2011 by John Wiley & Sons, Inc.

Keywords: DNA content; DNA synthesis; Click‐iT assay; surface and intracellular markers

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • 10 mM EdU (5‐ethynyl‐2′‐deoxyuridine; Invitrogen, cat. no. A10044) dissolved in DMSO; store up to 1 year at −20°C
  • Primary cells isolated from fresh tissue or cells cultured in appropriate cell culture medium
  • 10 mg/ml EdU dissolved in 0.9% NaCl (prepare fresh)
  • Phosphate‐buffered saline, without calcium and magnesium (CMF‐PBS), pH 7.2
  • Zinc salt‐based fixation buffer (ZBFB; see recipe)
  • Glycerol
  • Tris‐buffered saline (TBS), pH 7.4
  • Permeabilization buffer (see recipe)
  • Click‐iT EdU Alexa Fluor 488 Flow Cytometry Assay kit (Invitrogen, cat. no. 35002)
  • Antibodies:
    • PC7‐conjugated CD4 (Beckman Coulter, cat. no. 737660)
    • PE‐conjugated CD8 (DAKO, cat. no. R0806)
    • FITC‐conjugated CD14 (DAKO, cat. no. F0844)
    • APC‐conjugated CD45 (DAKO, cat. no. C7230)
    • PE‐Cy7‐conjugated Ly‐6A/E (Sca‐1; BD Pharmingen, cat. no. 558162)
    • AlexaFluor 647‐conjugated CD34 (eBiosciences, cat. no. 51‐0341‐82)
    • Mouse monoclonal antikeratin 10 antibody (LHP2, a generous gift from I. Leigh)
    • AlexaFluor 700 goat anti–mouse IgG (H+L) (Molecular Probes, cat. no. A21036)
  • Wash buffer (see recipe)
  • Ice
  • Hoechst staining solution (see recipe)
  • 37°C CO 2 incubator
  • 0.5‐ml Myjector U‐100 insulin syringes, 27‐G needle (BD Biosciences)
  • Centrifuge
  • Vortex
  • 4°C incubator
  • 2‐ml microcentrifuge tubes
  • Rocking table
  • Flow cytometer equipped with three lasers: Near‐UV 375 nm, blue 488 nm, and red 633 nm
NOTE: All protocols using live animals must first be reviewed and approved by an Institutional Animal Care and Use Committee (IACUC) and must follow officially approved procedures for the care and use of laboratory animals.
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Literature Cited

Literature Cited
   Beckstead, J.H. 1994. A simple technique for preservation of fixation‐sensitive antigens in paraffin‐embedded tissues. J. Histochem. Cytochem. 42:1127‐1134.
   Cappella, P., Gasparri, F., Pulici, M., and 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 A 73:626‐636.
   Cattoretti, G., Pileri, S., Parravicini, C., Becker, M.H., Poggi, S., Bifulco, C., Key, G., D'Amato, L., Sabattini, E., Feudale, E., Reynolds, F., Gerdes, J., and Rilke, F. 1993. Antigen unmasking on formalin‐fixed, paraffin‐embedded tissue sections. J. Pathol. 171:83‐98.
   Gillio‐Tos, A., De Marco, L., Fiano, V., Garcia‐Bragado, F., Dikshit, R., Boffetta, P., and Merletti, F. 2007. Efficient DNA extraction from 25‐year‐old paraffin‐embedded tissues: Study of 365 samples. Pathology 39:345‐348.
   Hicks, D.J., Johnson, L., Mitchell, S.M., Gough, J., Cooley, W.A., La Ragione, R.M., Spencer, Y.I., and Wangoo, A. 2006. Evaluation of zinc salt based fixatives for preserving antigenic determinants for immunohistochemical demonstration of murine immune system cell markers. Biotech. Histochem. 81:23‐30.
   Jensen, U.B., Owens, D.M., Pedersen, S., and Christensen, R. 2010. Zinc fixation preserves flow cytometry scatter and fluorescence parameters and allows simultaneous analysis of DNA content and synthesis, and intracellular and surface epitopes. Cytometry A 77:798‐804.
   Lykidis, D., Van Noorden, S., Armstrong, A., Spencer‐Dene, B., Li, J., Zhuang, Z., and Stamp, G.W. 2007. Novel zinc‐based fixative for high quality DNA, RNA and protein analysis. Nucleic Acids Res. 35:e85.
   Mason, J.T. and O'Leary, T.J. 1991. Effects of formaldehyde fixation on protein secondary structure: A calorimetric and infrared spectroscopic investigation. J. Histochem. Cytochem. 39:225‐229.
   Rousselle, C., Robert‐Nicoud, M., and Ronot, X. 1998. Flow cytometric analysis of DNA content of living and fixed cells: A comparative study using various fixatives. Histochem. J. 30:773‐781.
   Selevsek, N., Rival, S., Tholey, A., Heinzle, E., Heinz, U., Hemmingsen, L., and Adolph, H.W. 2009. Zinc ion‐induced domain organization in metallo‐beta‐lactamases: A flexible “zinc arm” for rapid metal ion transfer? J. Biol. Chem. 284:16419‐16431.
   Tang, X., Falls, D.L., Li, X., Lane, T., and Luskin, M.B. 2007. Antigen‐retrieval procedure for bromodeoxyuridine immunolabeling with concurrent labeling of nuclear DNA an antigens damaged by HCl pretreatment. J. Neurosci. 27:5837‐5844.
   Wester, K., Asplund, A., Backvall, H., Micke, P., Derveniece, A., Hartmane, I., Malmstrom, P.U., and Ponten, F. 2003. Zinc‐based fixative improves preservation of genomic DNA and proteins in histoprocessing of human tissues. Lab. Invest. 83:889‐899.
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