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Updated: Jul 27, 2025
Quantification of Reactive Oxygen Species Using 2′,7′-Dichlorofluorescein Diacetate Probe and Flow-Cytometry in Müller Glial Cells
Published on: May 13, 2022
Cell-type-specific determination of reactive oxygen species by flow cytometry
Agnes Hajnal1, Noemi Bogdandi1, Judit Marokházi1
1LabMagister Training and Science Ltd., Budapest, Hungary.
Background:
Seminal leukocyte-generated reactive oxygen species may have a significant impact on sperm intracellular reactive oxygen species levels, therefore contributing to oxidative damage and consequent functional impairment of spermatozoa. This relationship may be utilized for male urogenital inflammation-driven oxidative stress diagnostics.
Objective:
To obtain seminal cell-specific, reactive oxygen species-related fluorescence intensity cut-off values to differentiate leukocytospermic samples displaying reactive oxygen species overproduction (oxidative burst) from normozoospermic seminal samples.
Material And Methods:
Ejaculates gained by masturbation were obtained from patients in the framework of andrology consultations. The results published in this paper were generated from samples for which the attending physician requested spermatograms and seminal reactive oxygen species laboratory tests. Routine seminal analyses were performed according to World Health Organization guidelines. Samples were divided into normozoospermic "non-inflamed," and leukocytospermic groups. The semen was stained by 2',7'-dichlorodihydrofluorescein diacetate and the reactive oxygen species-related fluorescence signal and the percentage of reactive oxygen species-positive spermatozoa within the living population were quantified by flow cytometry.
Results:
Reactive oxygen species-related mean fluorescence intensity was higher in both spermatozoa and leukocytes from leukocytospermic samples than in those from normozoospermic samples. Mean fluorescence intensity in spermatozoa was positively and linearly correlated with mean fluorescence intensity measured in leukocytes in both groups.
Discussion:
The capacity of spermatozoa to generate reactive oxygen species is at least three log lower than that of granulocytes. The question is whether the reactive oxygen species-producing machinery of spermatozoa is capable of causing autologous oxidative stress or whether leukocytes are the predominant source of seminal oxidative stress. Based on our observations, the reactive oxygen species production of leukocytes may have a significant impact on the overall reactive oxygen species levels measured in spermatozoa.
Conclusion:
Reactive oxygen species-overproducing leukocytospermic and normozoospermic seminal samples can reliably be differentiated based on reactive oxygen species mean fluorescence intensity measurement.
