Flow acetone-staining technique: a highly efficient procedure for the simultaneous analysis of DNA content, cell morphology, and immunophenotype by flow cytometry

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
Maurizio CarbonariMassimo Fiorilli

Abstract

The accurate determination of cell cycle, immunophenotypes and morphology at single-cell level is not fully achieved by current flow cytometry protocols. Acetone, a coagulant fixative/permealizing agent, is widely used in static cytometry, but is impractical in flow cytometry because of its shrinking effect. We sought for conditions of acetone treatment that could permit the simultaneous analysis of physical parameters, surface and intracellular immunostaining, and DNA content. We evaluated different experimental conditions (concentration, duration of fixation, temperature, presence of proteins) to test the capacity of acetone fixation/permeabilization to preserve cell physical parameters (forward and side scatters, FSC, and SSC) and immunophenotyping while allowing stoichiometric DNA staining. The commonly used ethanol fixation technique was used as reference method. To detect phenotypes and DNA content simultaneously, we employed 7-aminoactinomycin D (7-AAD) as "intercalating" dye for DNA in spite of, or just for, its controversial ability in stoichiometric DNA staining. Cells were resting peripheral blood monucleated cells (PBMCs), T- and B-cell blasts obtained by PBMCs stimulation, and the human cell lines Ramos and Shep. A...Continue Reading

References

Nov 1, 1978·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·J FriedB D Clarkson
Jan 1, 1990·Methods in Cell Biology·L Vindeløv, I J Christensen
Apr 1, 1984·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·M TanakaE Ishikawa
Jun 3, 1994·Journal of Immunological Methods·W G TelfordP J Fraker
Jan 7, 2000·The Journal of Immunology : Official Journal of the American Association of Immunologists·G Hartmann, A M Krieg
Feb 1, 2000·Journal of Cellular Physiology·T Scholzen, J Gerdes

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Citations

Apr 27, 2010·PLoS Computational Biology·M Rowan BrownRachel J Errington
Feb 13, 2016·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Maurizio Carbonari
Jun 16, 2010·Acta histochemica·Sofie BreugelmansPaul Simoens
Dec 17, 2015·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Prashant TembhareP G Subramanian
Feb 4, 2011·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·M Rowan BrownRachel J Errington
Aug 16, 2018·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Giovanna RadicchioMaurizio Carbonari

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