Bivariate analysis of cellular DNA versus RNA content by laser scanning cytometry using the product of signal subtraction (differential fluorescence)as a separate parameter

Background: The cytometric methods of bivariate analysis of cellular RNA ve rsus DNA content have limitations. The method based oil the use of metachro matic fluorochrome acridine orange (AO) requires rigorous conditions of the equilibrium staining whereas pyronin Y and Hoechst 33342 necessitate the u se of an instrument that provides two-laser excitation, including the ultra violet (UNO light wavelength. Methods: Phytohemagglutinin (PHA)-stimulated human lymphocytes were deposit ed on microscope slides and fixed. DNA and double-stranded (ds) RNA were st ained with propidium iodide (PI) and protein was stained with BODIPY 630/65 0-X or fluorescein isothiocyanate (FITC). Cellular fluorescence was measure d with a laser scanning cytometer (LSC). The cells were treated with RNase A and their fluorescence was measured again. The file-merge feature of the LSC was used to record the cell PI fluorescence measurements prior to and a fter the RNase treatment in list mode, as a single file. The integrated PI fluorescence intensity of each cell after RNase treatment was subtracted fr om the fluorescence intensity of the same cell measured prior to RNase trea tment. This RNase-specific differential value of fluorescence (differential fluorescence [DF]) was plotted against the Cell fluorescence measured afte r RNase treatment or against the protein-associated BODIPY 630/650-X or FIT C fluorescence. Results: The scattergrams were characteristic of the RNA versus DNA bivaria te distributions where DF represented cellular ds RNA content and fluoresce nce intensity of the RNase-treated cells, their DNA content. The distributi ons were used to correlate cellular ds RNA content with the cell cycle posi tion or with protein content. Conclusions: One advantage of this novel approach based on the recording an d plotting of DF is that only the RNase -specific fraction of cell fluoresc ence is measured with no contribution of nonspecific components (e.g., due to the emission spectrum overlap or stainability of other than RNA cell con stituents). Another advantage is the method's simplicity, which ensues from the use of a single dye, the same illumination, and the same emission wave length detection sensor for measurement of both DNA and ds RNA. The method can be extended for multiparameter analysis of cell populations stained wit h other fluorochromes of the same-wavelength emission but targeted (e.g., i mmunocytochemically) for different cell constituents. Cytometry 45:73-78, 2 001. (C) 2001 Wiley-Liss, Inc.