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Three intravenous injections (1mg each) of biotin-X-NHS (BXN) given at 24h intervals labeled all circulating erythrocytes with biotin in C57Bl/6 mice. After 5 days, administration of another i.v. injection of BXN (0.6mg) resulted in the labeling of erythrocytes released in blood circulation after the first biotinylation step, with a lower intensity of biotin. The older erythrocyte population with high intensity of biotin (biotin^high population) and the later population of newly formed erythrocytes with lower intensity of biotin (biotin^low population) could be stained with streptavidin-APC (SAv) and identified by flow cytometry. Using the double biotinylation technique, we could examine the survival and age related changes in biotin^low population of erythrocytes that was released in circulation during a defined time period (5 days). Our results indicate that the percentage of Biotin^low erythrocytes in circulation remained static for 10 days …