A Double in vivo Biotinylation Technique for Objective Assessment of Aging and Clearance of Mouse Erythrocytes in Blood Circulation

Abstract

We have recently developed a new technique to objectively identify erythrocyte cohorts of defined age in mouse blood. The technique (termed double in vivo biotinylation, DIB) involves an initial biotinylation of all erythrocytes in circulation, followed after a few days by a second biotinylation, at a lower density, that labels the biotin-negative erythrocytes that have entered since the first biotinylation. The proportions of biotin^high, biotin^low, and biotin^negative erythrocytes are enumerated by flow cytometry. The DIB technique allows us to track age-related changes on erythrocyte cohorts (Protocol A), and to simultaneously identify very young and older erythrocyte populations in the blood (Protocol B). Using this technique, we have reexamined: i) the relationship between age and buoyant density of erythrocytes, ii) erythrocyte destruction through a random removal mechanism, and iii) the expression of phosphatidylserine on aging erythrocytes. We have also used the DIB technique to study age-related changes in the expression of various markers like CD47 and CD147 and green autofluorescence in aging erythrocyte populations.

Identification of Stages of Erythroid Differentiation in Bone Marrow and Erythrocyte Subpopulations in Blood Circulation that Are Preferentially Lost in Autoimmune Hemolytic Anemia in Mouse

“…Biotin negative erythrocytes in circulation would represent fresh and youngest erythrocytes released in blood after the second biotinylation step. Biotin low erythrocytes would represent the cohort of erythrocytes released in circulation between the first and the second biotinylation steps, and biotin high erythrocytes would represent the population of old residual erythrocytes that were present in blood at the time of first biotinylation step [18]. The schedule of biotinylation was fixed so that at the intended time of analysis (i.e., after 5–6 injections) the circulating erythrocytes comprise a very young cohort of biotin negative erythrocytes (less than 6 or 13 days old, depending upon the day of sacrifice) and a very old cohort of biotin high erythrocytes (more than 36 or 43 days old, depending upon the day of sacrifice) along with an intermediate aged biotin low erythrocyte cohort.…”

“…Biotin-labeled erythrocytes (1×10 6 ) were stained ex vivo with streptavidin-APC and anti-mouse CD71-PE/FITC in dark for 30 minutes to identify the different age cohorts of erythrocytes, as described before [13,18]. To enumerate the level of membrane-bound autoantibody in different subpopulations of circulating erythrocytes, cells (1×10 6 ) were co-stained with anti-mouse IgG/IgM-FITC along with streptavidin-APC and anti-mouse CD71-PE.…”

MtDNA mutagenesis impairs elimination of mitochondria during erythroid maturation leading to enhanced erythrocyte destruction

“…Survival of erythrocytes in vivo was determined using in vivo biotinylation of erythrocytes and analysis of lifespan of the biotinylated population by FACS analysis, exactly as described in ref. 53. In short, mice were injected intravenously with 1 mg of biotin (biotin-X-NHS, Merck Millipore) for 3 consecutive days, and 0.6 mg of biotin on the 8th day.…”

Elimination of Young Erythrocytes from Blood Circulation and Altered Erythropoietic Patterns during Paraquat Induced Anemic Phase in Mice

“…Biotin labeled erythrocytes were stained with streptavidin-FITC or streptavidin-APC and analyzed on a flow cytometer as described before [24].…”