DEVELOPMENT OF A PROPIDIUM IODIDE FLUORESCENCE ASSAY FOR PROLIFERATION AND CYTOTOXICITY ASSAYS

A propidium iodide fluorescence assay (PIA) was developed to character ize the in vitro growth of human tumor cell lines as well as to test t he cytotoxic activity of standard compounds. Propidium iodide (PI) was used as a dye which penetrates only damaged cellular membranes. Inter calation complexes are formed by PI with double-stranded DNA which eff ect an amplification of the fluorescence. Incubation of the total cell population with PI end subsequent fluorescence detection allowed asse ssment of the number of non-vital cells (first measurement). After fre ezing the cells at -20 degrees C for 24 h PI had access to total DNA l eading to total cell population counts (second measurement). The numbe r of viable cells was calculated by the difference between these two m easurements, In the proliferation and cytotoxicity assays 5 x 10(3) ce lls per well were plated in 96 multiwells and finally stained with 50 mu g/ml PI in 25 mu l for 10 min. A correlation between the log of cel l 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 1 50-500 cells/wells. In cytotoxicity assays eight clinically used cytos tatics were tested which effected a clear dose-response relationship ( r = 0.93-0.98) and high reproducibility (r = 0.92). In conclusion, thi s 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 nece ssary for drug testing. The PIA can easily be used for cell number det erminations in biological and pharmacological studies.