FLOW CYTOMETRIC ANALYSIS OF MICRONUCLEATED RETICULOCYTES IN MOUSE BONE-MARROW

This laboratory has previously reported a flow cytometric procedure fo r quantitatively analyzing mouse peripheral blood reticulocytes for mi cronucleus content. The current study extends this line of investigati on by evaluating whether these same flow cytometric scaring procedures can be applied to the analysis of mouse bone marrow samples. To valid ate the method, three groups of male BALB/c mice were treated with 100 mg/kg b.wt. methyl methanesulfonate. Bone marrow samples were collect ed 20, 40 or 60 h after administration. A set of 5 untreated animals w as included to provide an indication of spontaneous micronucleus frequ encies. The cells were fixed with ultracold methanol, treated with rib onuclease, and labeled with anti-CD71 antibody (FITC conjugate) and pr opidium iodide. This fixing and labeling procedure resulted in the res olution of the micronucleated reticulocyte population and facilitated high-speed acquisition and enumeration via flow cytometry, The number of micronucleated reticulocytes was determined flow cytometrically by the analysis of 10000 total reticulocytes per bone marrow sample. In a ddition to these automated measurements, slides stained with acridine orange were prepared and the number of micronuclei per 1000 reticulocy tes was determined microscopically for each sample. The resulting data demonstrate that flow cytometry can effectively enumerate micronuclea ted reticulocytes in mouse bone marrow. The advantages associated with an objective, high throughput scoring methodology are also clearly in dicated.