Simultaneous Three Color Detection of Surface Antigen (My 7), Intracellular Antigen (c-myc), and DNA Content using Single Laser Flow Cytometry

Han, Ku Taek; Ryu, Ki Sung; Namkoong, Sung Eun; Kim, Soo Pyung; Rha, Jong Gu; Song, Seung Kyu; Kim, Seong Jo; Lee, Hun Young; Parker, John

Abstract

Flow cytometry, a useful tool for measuring DNA content and cell differentiation as expressed by cell surface markers, is utilized to measure multiple antigens, especially surface antigen, intracellular oncoprotein, and DNA content, simultaneously. For this simultaneous detection, several methods off ixation and permeabilization have been used with limited values. In this study, 20 ng/ml of lysolecithin in 1% paraformaldehyde solution was utilized for fixation and permeabilization of cultured promyelocytic leukemic cells(HL 60). The cells were first stained with phycoerythrin (PE)-conjugated monoclonal antibody to the cell surface My 7 antigen and then were fixed and permeabilized with 20 ng/ml of lysolecithin in 1% partormaldehyde solution. After incubation, the fixed and permeabilized cells were stained with monoclonal antibody to intracellular c-myc antigen, which were followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibody. The c-myc stained cells were finally stained for DNA content with 7-amino-actinomycin D(7-AAD). This procedure permits excellent staining for intracellular oncoproteins and preservation of surface antigens with relatively low cofficients of variation (CV) for the G0G1 peak of the DNA histograms and suggests that the sequential staining procedure of surface antigen, intracellular antigen, and DNA content will be extended for the study of correlations with cellular differentiation, expression of oncoproteins, and cell cycle analysis in the cells which are obtained from human malignant diseases using a 488 nm single laser flow cytometry.