Flow acetone-staining technique: a highly efficient procedure for the simultaneous analysis of DNA content, cell...

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. Acetone fixation, preserving both the recovery and the physical parameters of cells, is drastically influenced by temperature of treatment and is practicable only when the protocol is realized at 8 degrees C. Under this condition, acetone maintains the immunophenotypic fluorescences (realized before or after the fixation) better than ethanol. Stoichiometric DNA staining of acetone processed cells, the variation coefficients (CV) of frequency distributions of G1/G0 and G2/M phases, the modes ratio of these distributions and doublets generation are at least comparable to those obtained with ethanol treatment. The assay developed in the present study, that we called flow acetone-staining technique (FAST), accurately analyzes cell cycle, physical parameters and immunophenotypes in heterogenous cell populations, and thus provides a useful tool for cytomics.