Appearance of the terminal senescent cell population in human diploid fibroblasts analyzed by flow cytometry

Abstract

We studied changes in the distribution pattern of relative RNA content during the in vitro aging of TIG-3 cells by flow cytometry (FACS III). Propidium iodide (PI) does not stain total cellular RNA, but it intercalates specifically into double-helical regions of both DNA and RNA. In applying this principle to RNA, we stained double-stranded RNA (dsRNA) in whole cells with PI after DNA digestion with DNase. The results showed that dsRNA distribution patterns were relatively constant at 7–75 population doublings (PD) but were significantly altered after 77 PD. The distribution patterns were similar as those for cell volume measured with a Coulter Counter. The total cellular dsRNA contents increased linearly at the senescent phase of their in vitro life span. In contrast, the mean dsRNA contents (50% dsRNA contents) rapidly increased to 77–79 PD, but decreased somewhat at 81–83 PD. Two-dimensional histograms of the dsRNA contents versus cell size were little altered from 25 PD to 75 PD. However, a population with relatively larger cell volume and weaker fluorescence intensity appeared and increased after 79 PD. This cell population group may be categorized as “terminal senescent cells” that no more divide in respect that the dsRNA content decreases in spite of the increase of total RNA content.