Immunophenotyping and DNA Content Analysis of Acetone‐Fixed Cells

Debora Mancaniello1, Maurizio Carbonari1

1 Laboratory of Immunology, Clinical Medicine Department, University of Rome “La Sapienza,”, Rome, Italy
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 9.26
DOI:  10.1002/0471142956.cy0926s46
Online Posting Date:  October, 2008
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The flow acetone staining technique (FAST) allows one to concurrently study physical cell features revealed by light‐scatter analysis, surface/nuclear phenotypes, and cellular DNA content. Thus, diverse subpopulations of proliferating cells can be identified in heterogeneous populations by their immunophenotype and their cell cycle status, and DNA ploidy can be assessed. Acetone, a coagulant (precipitating) fixative that also has the ability to permeabilize cell membranes, is widely used in static cytometry, but rarely in flow cytometry because of its undesirable effects, namely causing cell shrinkage. Nevertheless, when employed under proper temperature conditions (∼8°C), it preserves cellular physical features and immunophenotype well, and is compatible with stoichiometric DNA staining and accurate measurement of DNA content. Due to these virtues of FAST, the method provides useful approaches for cell biology and hematology/oncology studies. Curr. Protocol. Cytom. 46:9.26.1‐9.26.11. © 2008 by John Wiley & Sons, Inc.

Keywords: acetone; multiparametric flow cytometry; DNA content; conservative fixation; proliferating cells

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Table of Contents

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

  • Proliferating cells
  • Fluorescein isothiocyanate (FITC), phycoerythrin (PE), and allophycocyanin (APC) conjugated monoclonal antibodies (MoAbs) against surface and cytoplasmic phenotypes (e.g., CD3, CD8, CD4, CD19, anti‐κ chain, anti‐λ chain, anti–human IgM) and nuclear antigens (e.g., PCNA, ki67)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Fetal bovine serum (FBS; appendix 2A)
  • Acetone (phenol free, ≥99.5%)
  • 7‐aminoactinomycin D (7‐AAD)
  • Washing/staining (w/s) solution (see recipe)
  • 1.5‐ml polypropylene tubes
  • Refrigerated centrifuge
  • Refrigerator for incubation at 8°C
  • Flow cytometer with a 488 nm argon‐ion laser (and eventually with a 635 nm red laser) as fluorescence excitation source
  • Nile Red beads and APC beads (Becton Dickinson)
  • 12 × 75–mm polystyrene tubes
  • Software to deconvolute cellular DNA content frequency histograms (e.g., ModFit from Verity Software House; or MultiCycle from Phoenix Flow Systems)
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Literature Cited

Literature Cited
   Darzynkiewicz, Z., Juan, G., and Bedner, E. 1999. Determining cell cycle stages by flow cytometry. Curr. Protoc. Cell Biol. 1:8.4.1‐8.4.18.
   Eudey, T.L. 1996. Statistical considerations in DNA flow cytometry. Stat. Sci. 11:320‐324.
   Hudson, L. and Hay, F.C. 1989. Practical Immunology, 3rd. ed. Blackwell Scientific Publications, Oxford, U.K.
   Lanuti, P., Marchisio, M., Cantilena, S., Paludi, M., Bascelli, A., Gaspari, A.R., Grifone, G., Centurione, M.A., Papa, S., Di Pietro, R., Cataldi, A., Miscia, S., and Bertagnolo, V. 2006. A flow cytometry procedure for simultaneous characterization of cell DNA content and expression of intracellular protein kinase C‐ζ. Immunol. Meth. 315:37‐48.
   Rabinovitch, P.S., Torres, R.M., and Engel, D. 1986. Simultaneous cell cycle analysis and two‐color surface immunofluorescence using 7‐amino‐actinomycin D and single laser excitation: applications to study of cell activation and the cell cycle of murine ly‐1 B cells. J. Immunol. 136:2769‐2775.
   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.
   Sato, Y., Mukai, K., Watanabe, S., Goto, M., and Shimosato, Y. 1986. The amex method. A semplified technique of tissue processing and paraffin embedding with improved preservation of antigens for immunostaining. Am. J. Pathol. 125:431‐435.
   Schimenti, K.J. and Jacobberger, J.W. 1992. Fixation of mammalian cells for flow cytometric evaluation of DNA content and nuclear immunofluorescence. Cytometry 13:48‐59.
   Tanaka, M., Tanaka, H., and Ishikawa, E. 1984. Immunohistochemical demonstration of surface antigen of human lymphocytes with monoclonal antibody in acetone‐fixed paraffin‐embedded sections. J. Histochem. Cytochem. 32:452‐454.
   Telford, G.W., King, L.E., and Fraker, P.J. 1994. Rapid quantitation of apoptosis in pure and heterogeneous cell populations using flow cytometry. J. Immunol. Meth. 172:1‐16.
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