CELL KINETIC-STUDIES IN MOUSE TONGUE MUCOSA BY AUTORADIOGRAPHIC, IMMUNOHISTOCHEMICAL AND FLOW CYTOMETRIC TECHNIQUES

A number of techniques, including autoradiography after in vivo admini stration of tritiated thymidine ([H-3]dT), immunohistochemistry after in vivo administration of bromodeoxyuridine (BrdUrd), and flow cytomet ry (FCM) with and without BrdUrd detection were compared in the epithe lium of ventral mouse tongue. Investigation of the diurnal proliferati ve rhythm by immunohistochemical detection of incorporated BrdUrd with different primary antibodies in combination with the alkaline-phospha tase-anti-alkaline-phosphatase technique, the peroxidase-anti-peroxida se method, and an indirect method with a polyclonal peroxidase-conjuga ted secondary antibody yielded results similar to standard autoradiogr aphy. Preparation of single cell suspensions for flow cytometry was no t successful. A maximum yield of about 8.5% of the original cell numbe r was achieved by ultrasound disintegration in combination with trypsi n and dithioerythrol treatment, but neither a G(0)/G(1) peak nor a G(2 ) + M peak was observed in DNA histograms. A better yield of about 38% of the original nuclei number was obtained by preparation of suspensi ons of nuclei using citric acid and the detergent Tween 20 in combinat ion with magnetic stirring. Both S-phase index and BrdUrd labelling in dex could be determined by FCM and showed the normal diurnal variation s. However, the BrdUrd labelling index in suspensions of nuclei was si gnificantly higher than the labelling index determined after immunohis tochemistry. The FCM S-phase index at times of day with low DNA synthe sizing activity was higher than the BrdUrd index, indicating a fractio n of unlabelled S-phase cells. In conclusion, detection of incorporate d BrdUrd in oral mucosa by immunohistochemical techniques or flow cyto metry is feasible and provides a useful tool for fast measurements of proliferation.