IN-VITRO BROMODEOXYURIDINE-LABELING OF SINGLE-CELL SUSPENSIONS - EFFECTS OF TIME AND TEMPERATURE OF SAMPLE STORAGE

The present study was aimed to explore how the in vitro BrdUrd-labelli ng of rat thymocytes might be affected by both the time elapsed betwee n obtaining the sample and the beginning of the labelling (0, 15, 30 o r 60 min) and the effect of the temperature of storage (4 degrees C ve rsus room temperature). Single cell suspensions obtained after in vivo labelling with BrdUrd were used as controls. The S phase fraction was calculated by flow cytometry both according to BrdUrd-immunolabelling and DNA content. Immediate incubation with BrdUrd after the sample wa s obtained resulted in a slight decrease of the proportion of S phase cells analysed either according to DNA content or to BrdUrd-immunolabe lling. Regardless of storage-temperature, the S phase fraction decreas ed in samples kept for 15 min or more before BrdUrd incubation. No Brd Urd-positive cells were detected in samples stored for 60 min at room temperature. This effect was related to temperature since positive cel ls were found when the samples were kept at 4 degrees C during the sam e time period. Our results suggest that during in vitro incubation a r elative loss of S phase cells exists and that a delay beyond 15 min be tween obtaining the sample and the in vitro labelling seriusly comprom ises the results of this technique.