Development of a propidium iodide fluorescence assay for proliferation and cytotoxicity assays


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Abstract

A propidium iodide fluorescence assay (PIA) was developed to characterize the in vitro growth of human tumor cell lines as well as to test the cytotoxic activity of standard compounds. Propidium lodide (PI) was used as a dye which penetrates only damaged cellular membranes. Intercalation complexes are formed by PI with double-stranded DNA which effect an amplification of the fluorescence. Incubation of the total cell population with PI and subsequent fluorescence detection allowed assessment of the number of non-vital cells (first measurement). After freezing the cells at −20°C for 24 h PI had access to total DNA leading to total cell population counts (second measurement). The number of viable cells was calculated by the difference between these two measurements. In the proliferation and cytotoxicity assays 5 x 103 cells per well were plated in 96 multiwells and finally stained with 50 μg/ml PI in 25 μl for 10 min. A correlation between the log of cell number and the log of flurorescence units could be demonstrated over a 2.5–3 log range (r = 0.97). The lower limit of cell detection was 150–500 cells/wells. In cytotoxicity assays eight clinically used cytostatics were tested which effected a clear dose-response relationship (r = 0.93–0.98) and high reproducibility (r = 0.92). In conclusion, this assay is a simple and rapid test system, the main advantages are the absence of any washing steps and the small number of tumor cells necessary for drug testing. The PIA can easily be used for cell number determinations in biological and pharmacological studies.

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