Evaluation of eight fluorochrome combinations for simultaneous DNA-protein flow analyses.
Eight fluorescent dye combinations for simultaneous DNA-protein staining have been evaluated spectroscopically and flow microfluorometrically: propidium iodide (PI) with fluoresceinisothiocyanate (FITC), fluorescamine (FC), and dansylchloride (DANS); diamidinophenylindole (DAPII) with sulphorhodamin (SR101), tetramethylrhodamin isothiocyanate (TRITC), and nitrobenzodiazole (NBD); acriflavine (AF) with stilbene isothiocyanate sulphonic acid (SITS), and DAPI. Three different experimental tumor cell lines have been employed in the investigations. Simultaneous DNA-protein analyses have been carried out with the newly developed HEIFAS instrument. Spectroscopically two groups of dyes were distinguishable according to their excitation maximum below 400 nm and above 450 nm respectively. DANS and NBD were found to be unsatisfactory with respect to their protein distributions obtained by flow analysis. The remaining stains involved in the dye combination revealed comparable flow distributions of the cellular DNA and protein content. With respect to preparation time and number of centrifugal steps involved in the staining protocols, and in connection with the stability of the dye used, the DAPI-SR101 method proved to be fastest and easiest. With this combination DNA and protein flow analysis can be performed simultaneously within 30 min.
D000818:Animals; D002279:Carcinoma 256, Walker / Q000032:analysis; D003584:Cytological Techniques; D004273:DNA, Neoplasm / Q000032:analysis*; D005456:Fluorescent Dyes; D008114:Liver Neoplasms, Experimental / Q000032:analysis; D009363:Neoplasm Proteins / Q000032:analysis*; D009374:Neoplasms, Experimental / Q000032:analysis*; D051381:Rats; D012517:Sarcoma, Yoshida / Q000032:analysis; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling
D004273:DNA, Neoplasm; D005456:Fluorescent Dyes; D009363:Neoplasm Proteins