A unified procedure for conservative (morphology) and integral (DNA and immunophenotype) cell staining for flow cytometry.
BACKGROUND
Current methods for multiparameter DNA flow cytometry suffer from several limitations. These include significant modifications of cell morphological parameters, the impossibility to counterstain cells with certain fluorochromes, and laborious tuning of the instrument that, for some procedures, must be equipped with an ultraviolet (UV) laser. To overcome these problems, we developed a novel method for the simultaneous analysis of morphological parameters, four-color immunophenotyping, and stoichiometric DNA labeling using a bench-top flow cytometer.


METHODS
The method consists of a mild permeabilization/fixation treatment at room temperature, followed by labeling with fluorochrome-conjugated monoclonal antibodies (mAbs) and with the DNA dye 7-aminoactinomycin D (7-AAD) at 56 degrees C.


RESULTS
Using this method, we analyzed resting peripheral blood mononucleated cells (PBMC), proliferating T cells cultured in the presence of interleukin-2 (IL-2), and lymphoblastoid B cells. Lymphocytes, monocytes, and lymphoblasts treated by this procedure retained differential light scattering (DLS) characteristics virtually identical to those of untreated cells. This allowed regions to be drawn on forward scatter (FSC) and side scatter (SSC) cytograms resolving different cell populations. DLS were preserved well enough to distinguish large lymphoblasts in the S or G2/M phases from small G0/G1 cells. Also, stainability with fluorescein-isothiocyanate (FITC), R-phycoerythrin (PE), allophycocyanin (APC)-conjugated mAbs was generally preserved. DNA labeling with 7-AAD was of quality good enough to permit accurate cell cycle analysis.


CONCLUSIONS
The method described here, which we called integral hot staining (IHS), represents a very simple, reproducible, and conservative assay for multiparameter DNA analysis using a bench-top flow cytometer. Last but not least, the cytometer tuning for multiparameter acquisition is straightforward.
Carbonari
M
M
Department of Clinical Medicine, University of Rome "La Sapienza," Rome, Italy.
Tedesco
T
T
Fiorilli
M
M
D001402:B-Lymphocytes / Q000166:cytology; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D016130:Immunophenotyping; D007963:Leukocytes, Mononuclear / Q000166:cytology; D010798:Phycocyanin; D010799:Phycoerythrin; D013194:Staining and Labeling / Q000379:methods*
C002497:allophycocyanin; D010798:Phycocyanin; D010799:Phycoerythrin; D004247:DNA; D016650:Fluorescein-5-isothiocyanate