Flow cytometric analysis of blood cells stained with the cyanine dye DiOC1[3]: Reticulocyte quantification

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Abstract
The fluorescent dye 3,3′‐dimethyloxacarbocyanine (DiOC1[3]) is taken up by all cells in mammalian blood which then fluoresce as follows: mature erythrocytes < immature erythrocytes ≃ platelets < leukocytes. A continuous fluorescence distribution can be generated for the red blood cells by flow cytometry and deconvolved into two arbitrary populations, mature and immature erythrocytes (mRBC and imRBC). This analysis mimics the established method of counting imRBC stained with the supravital dyes, new methylene blue, brilliant cresyl blue (BCB), and acridine orange (AO). However, the population of imRBC as quantified by DiOC1[3] flurorescence is a subset of reticulocytes (reticulocytes as determined by BCB assay). The advantages and disadvantages of using DiOC1[3], AO, or pyronine Y as reticulocyte stains are discussed.