Flow cytometric assessment of homeostatic aging of reticulocytes in rats

Abstract

Objective: The purpose of this work was to develop a flow cytometry method of obtaining RNA-thiazole orange signal distribution of reticulocytes (RET) from bone marrow aspirates and determine the distribution of age for RET in the blood and bone marrow.

Methods: The thiazole orange (an RNA dye) and LDS-751 (a DNA dye) composition was used to separate RET from other cell populations. LDS 751 was used to separate red blood cells (RBC) from nucleated cells. The differentiation between mature RBC and RET was done by means of the presence of residual RNA. It also served as a marker of age of RET. In vitro changes of the fluorescence signal of the RET population allowed us to obtain the unique relationship between the signal and the age for an individual cell and was used to transform the in vivo signal distribution into the age distribution.

Results: The determination of age was not possible due to the lack of knowledge of the RET signal distribution at birth. Instead, a relative time (RT) that a RET needs to become a mature RBC was used. The RT distribution of RET in blood and bone marrow was determined. The RT that an average RET spends in the blood is 0.43 days and ranges from 0 to 1.1 days. The time an average RET in bone marrow needs to become a mature RBC, is 1.36 days. The RET born with the highest signal needs about 1.8 days to become a mature RBC. About 37% reticulocytes die in the circulation.

Conclusions: In summary, the thiazole orange staining can be used for determination of the RET age distribution. However, only the RT distribution can be determined. The proposed method was successfully applied to characterize in vivo aging of blood and bone marrow RET.