Abstract
Measurement of autoradiographic grains produced by the decay of incorporated radioisotopes is often used for a quantitative assay of the rate of DNA replication and DNA repair in cells or tissues. However, visual grain counting by microscopic observation is time-consuming and tedious process. Recently, Kraemer et al. reported that automated measurement of grains in cultured human cells may be facilitated by using appropriate grain counting instruments. Under their experimental conditions using Kodak NTB-3 emulsion, instrument-determined grain number per nucleus was proportional to visual counts up to 30 grains, and then leveled off at much larger visual counts. The saturation phenomenon was due to counting-loss by the instrument caused by overlapping of neighboring grains.
To prevent the counting-loss, we have used in the present study Japanese Sakura NR-M2 emulsion which is less sensitive to radiation exposure than Kodak NTB-3, thereby yielding smaller size of grains per radioactive decay. Samples were prepared from cultured skin fibroblasts derived from normal individuals and xeroderma pigmentosum (XP) patients defective in DNA repair. These cells were irradiated with 254 nm UV incubated for 3h with culture medium containing 3H-thymidine, and autoradiograms were made by dipping in Sakura NR-M2 emulsion. The number of grains as well as grain surface area per nucleus was measured by using ARTEK CYTO TALLY MODEL 900 counting instrument, and compared with visual counts. The results showed that, under our optimum condition, the instrument-determined number of grains was directly proportional to visual counts, at least up to 150 grains per nucleus, with a correlation coefficient of 0.971. The instrument-determined grain surface area was also linear in comparison to visual counts with the same correlation coefficient, 0.971. Automated analysis of grain numbers enabled us to estimate the rates of ultraviolet-induced unscheduled DNA synthesis (UDS) in XP cells; the instrument-determined UDS in a XP cell strain relative to that in normal cells was 10.0%, which was very close to the value obtained by visual counts (11.1%) . The time required for the instrumental counting was about one-sixth of that of visual counting.
