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Immunophenotypic and Morphometric Evaluation of Bone Marrow Macrophage Culture Stimulated by Sodium Aminodihydrophthalazinedione In Vitro

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Abstract

Morphometric and immunophenotypic characteristics of rat bone marrow macrophages isolated from intact animals and stimulated with sodium aminodihydrophthalazinedione (ADPN) under cultivation for 24, 48, and 72 h were studied. The following morphometric parameters were determined: the cell area, the area of the cytoplasm and nuclei, as well as the nuclear-cytoplasmic ratio (NCR). The proliferative activity was evaluated on the basis of the presence of the Ki-67 marker. The phenotype of macrophages was determined by the markers CD163 (M2 phenotype) and F4/80 (M1 phenotype) present on the cell surface. In addition, the content of the TGF-β growth factor in the macrophage cytoplasm was evaluated. The stimulation of macrophages with ADPN at a dose of 50 μg/mL promotes an increase in the number of CD163+ cells and the content of this marker with increasing cultivation time. On the contrary, the action of 100 μg/mL ADPN leads to an increase in the proportion of F4/80+ cells in the culture and to the growth in the content of this marker with increasing cultivation time. The accumulation of TGF-β occurs for 48 h of cell culturing in the presence of 50 μg/mL ADPN, and its increased content is retained for 72 h. ADPN at the dose of 100 μg/mL enhances the formation of TGF-β to 48 h and inhibits it to 72 h of cell culturing with the substance. The maturation of macrophages is accelerated when ADPN is added to the culture of monocytes, and the substance also has a pronounced dose-dependent effect on BM macrophages.

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Funding

This work was performed within the framework of the Budget Program “Study of the Mechanisms of Regenerative Processes in Organs and Tissues Using Experimental Models of Extreme Factors and Toxic Effects on the Body,” state registration no. АААА-А18-118020590107-0.

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Authors and Affiliations

  1. Institute of Immunology and Physiology, Ural Branch of the Russian Academy of Sciences, 620049, Yekaterinburg, Russia

    V. A. Pozdina & I. G. Danilova

  2. Ural State Medical University, 620028, Yekaterinburg, Russia

    U. V. Zvedeninova

  3. Institute of Natural Sciences and Mathematics, Ural Federal University, 620026, Yekaterinburg, Russia

    M. V. Ulitko

  4. Institute of Medical Cell Technologies, 620026, Yekaterinburg, Russia

    M. V. Ulitko & I. G. Danilova

  5. Institute of Immunology and Preventive Medicine, 1000, Ljubljana, Slovenia

    M. T. Abidov

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  1. V. A. Pozdina
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Correspondence to V. A. Pozdina.

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Conflict of interest. The authors declare that they have no conflicts of interest.

Statement on the welfare of animals. All applicable international, national, and institutional guidelines for the care and use of animals were followed. All experiments on animals were approved by the Ethics Committee of the Institute of Immunology and Physiology of the Ural Branch of the Russian Academy of Sciences (no. d-TM-2016-20) and performed in accordance with the principles formulated in Directive 2010/63/EU.

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Translated by D. Novikova

Abbreviations: BM—bone marrow, CCM—complete culture medium, EBI—erythroblastic island, NO—nitrogen oxide, HSC—hematopoietic stem cell.

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Pozdina, V.A., Zvedeninova, U.V., Ulitko, M.V. et al. Immunophenotypic and Morphometric Evaluation of Bone Marrow Macrophage Culture Stimulated by Sodium Aminodihydrophthalazinedione In Vitro. Cell Tiss. Biol. 15, 594–603 (2021). https://doi.org/10.1134/S1990519X21060158

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