Flow cytometric analysis of RNA content in different cell populations using pyronin Y and methyl green

Abstract

Pyronin Y (PY) was used, in flow cytometric (FCM) systems, to estimate the RNA content per cell in formalin fixed EL4 leukosis tumor cells, enzyme dispersed R3327-G rat prostatic adenocarcinoma cells, mouse spleen cells stimulated with concanavalin A, and human peripheral blood lymphocytes stimulated with phytohemagglutinin. Preincubation of the cells with methyl green (MG) blocked PY binding to DNA such that the intracellular fluorescence from MG-PY was due primarily to its binding to RNA. Treatment of the cells with ribonuclease resulted in a 3-to &fold reduction in the fluorescence intensity of intracellular MG-PY.Mitogen stimulation of either mouse or human lymphocytes resulted in an increase in DNA (propidium iodide fluorescence) and RNA (MG-PY fluorescence) content per cell over resting levels. Further, the changes in stimulated human lymphocyte DNA and RNA contents following 24,48, and 72 hr of cell culture were monitored, The results showed that RNA levels were significantly increased prior to that of DNA. Also, the effects of different cell cycle phase specific blocking agents on lymphocyte cell cycle traverse were investigated. We found that: a) actinomycin D inhibited the increases in cellular RNA and DNA; b) hydroxyurea inhibited the increases in cellular DNA whereas increases in cellular RNA were only slightly reduced; c) tritiated thymidine caused an accumulation of cells having high DNA and RNA contents; and d) Colcemid promoted an accumulation of cells having high DNA contents while causing a reduction of cells having high RNA contents. These results were nearly identical to reports by other investigators using the metachromatic dye acridine orange to quantitate RNA per cell. Thus, the MG-PY technique described is indicated to provide a stable and accurate measure of RNA content per cell.Key terms: Pyronin Y, methyl green, flow cytometry, RNA Over the last decade the automated identification and classification of cellular subpopulations has developed extensively using flow cytometric (FCM) instrumentation. This instrumentation has been particularly useful in the rapid discrimi-'

Application of pyronin Y(G) in cytochemistry of nucleic acids

“…In an accompanying paper (171, we describe biophysical and biochemical studies on pyronin Y (PY) and interactions of this dye with natural and synthetic nucleic acids in solutions. Several observations from these studies could be of importance for the qualitative and quantitative application of PY, both as an absorption dye for light microscopy (2,191 and as a fluorochrome (4,23,26,28). In the present paper, we demonstrate that the biophysical properties of the dye complexes with nucleic acids observed in solutions (17) are relevant in cytochemistry.…”

Grading melanocytic dysplasia in paraffin wax embedded tissue by the nucleic acid index

“…Previous studies have shown that total RNA values are raised as a function of the tumour proliferation rate. [20][21][22][23] Thus, the NAI provides a quantitative correlate for the proliferative potential of melanocytic lesions. Indeed, strong inverse correlations were found between NAI and both mitotic index and Breslow thickness.…”

Flow cytometry and RNA studies

“…DiO-CI(3) [21], thioflavin T [27], and thiazole orange [23] have been used for viable reticulocyte analysis. Pyronin Y (PY) has also been used for automatic counting of reticulocytes by Tanke et al [31], but this dye, specific for double-stranded RNA, is also used in association with other dyes like methyl green for RNA content analysis [26], or with Hoechst 33342 for RNA versus DNA analysis in unfixed cells [29]. One inconvenience of PY is that staining is not always stoichiometric, as PY seems to be sensitive to the conformation of the rRNA, and its fluorescence drops whefla polysomes dissociate [13].…”