We have recently reported an innovative approach to use charged fluorochromes such as propidium iodide (PI) in the real-time, dynamic cell viability assays. The present study was designed to provide a mechanistic rationale for the kinetic assays using cell permeability markers. Uptake of PI by live cells, effect on the cell cycle, long term proliferation capacity, DNA damage response and pharmacologic interactions with anticancer drugs were studied using both laser scanning microscopy and laser scanning cytometry. Exposure of human carcinomic alveolar basal epithelial A549 cells in cultures to 1.5 or 7.5 µM of PI for 24 h had minimal effect on cell cycle progression including DNA replication as measured by incorporation of 5’-ethynyl-2-deoxyuridine (EdU) detected by the “click chemistry” approach and measured by laser scanning cytometry. A modest reduction, from 44% to 40% or 33%, in frequency of DNA replicating cells was seen after 48 h at 1.5 or 7.5 µM concentration of PI. There was no evidence of increased phosphorylation of histone γH2AX in cells growing in the presence of 1.5 or 7.5 µM of PI for up to 48 h. Confocal image analysis, of HeLa and NIH 3T3 mouse embryonic fibroblasts growing in the presence of PI showed granular distribution in cell cytoplasm suggesting PI accumulation in endosomes and progressive increase in fluorescence of nucleoli reflecting PI binding to nucleolar RNA. The overall responses of cells to cytotoxic agents were also not affected by the growth in the presence PI. Our data lend further support to the notion that propidium iodide can be effectively used in real-time, kinetic viability assays.

Fluorescence recovery after photobleaching (FRAP) is a tool widely used in studies of dynamic behavior of fluorescently-tagged proteins in live cells. We have analyzed published data on dynamics of various nuclear proteins and note that FRAP protocols and methods of data analysis vary between laboratories. A question arises if the experimental protocol can influence the recovery times. To establish if the FRAP protocol can influence fluorescence half-recovery times, we used various FRAP protocols and studied the dynamics of a GFP-tagged H1 (linker) histone. We demonstrate that fluorescence half-recovery times depend on the bleaching protocol, including the photon flux of the bleaching light. Thus, we conclude that due to differences between protocols and ways of analyzing data, the existing body of information on mobility of various nuclear proteins does not permit direct comparisons between experiments from different laboratories. To exploit a full potential of FRAP as a quantitative technique, there is a need to establish ground rules for photobleaching protocols and adopt a consistent method of data analysis. ' 2010 International Society for Advancement of Cytometry Key termsFRAP; fluorescence recovery after photobleaching; nuclear proteins; histone; mobile fraction; protein dynamics FLUORESCENCE recovery after photobleaching (FRAP) is a quantitative technique capable of measuring rates of exchange of proteins at their binding sites in a cell, and proportions between their mobile and immobile fractions. With an intention of comparing the protocols and the dynamic behavior of various nuclear proteins, we analyzed 47 original papers describing FRAP studies (protein dynamics data and FRAP technical information extracted from these papers has been compiled into Supp. Info. Table 1), as well as nine theoretical and 43 review papers on the subject. A broad analysis was rendered difficult, as the description of FRAP protocols was often incomplete or even lacking. Only three papers provided a full description of their FRAP protocol and stated if correction for movements of the cell or the nucleus was required and applied. Only eight papers provided information about the pixel size, or experimental conditions that enabled the reader to calculate this parameter. In 27 papers there was no information about the area of the bleached region, 29 papers provided only a general description (half of the nucleus or a strip across the nucleus). Eleven papers had no information about intensities of the bleaching light, 16 papers gave no information about the objective lens.In principle, the lack of technical information need not prevent interexperiment analysis of protein dynamics data, so long as the fluorescence recovery times are independent of the bleaching protocol. To establish if recovery times are indeed independent of the bleaching conditions, we performed FRAP experiments using GFPtagged histone H1 in HeLa cells. Histone H1 is highly dynamic; it has a large bound and a smaller unbound fraction (1,2). Tagging H1 ...