Determination of the content of FAD cofactor and NAD(P)H-oxidase complexes in mouse splenocytes and Lewis carcinoma cells under conditions of apoptosis by confocal microscopy method

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Abstract

In this work, using fluorescence and confocal microscopy, we studied the content of the cofactor FAD and enzymatic NAD(P)H-oxidase complexes (with fluorophores AnnexinV-FITC, 7-AAD (7-aminoactinomycin D), EtBr) under conditions of apoptosis caused by sodium anphene with hydrogen peroxide in healthy mouse splenocytes and Lewis carcinoma tumor cells. The use of fluorescence microscopy allows observing and quantifying the apoptotic effect of sodium anphen and hydrogen peroxide, and visualization of metabolic changes in the cell, including increased fluorescence of FAD in tumor cells and NAD(P)H-oxidase complexes in splenocytes. The data obtained indicate the possibility of using sodium anphen in combination with hydrogen peroxide as an antitumor drug acting on certain types of cells.

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About the authors

E. M. Mil

Emanuel Institute of Biochemical Physics Russian Academy of Sciences

Email: albantovaaa@mail.ru
Russian Federation, Moscow

A. A. Albantova

Emanuel Institute of Biochemical Physics Russian Academy of Sciences

Author for correspondence.
Email: albantovaaa@mail.ru
Russian Federation, Moscow

L. I. Matienko

Emanuel Institute of Biochemical Physics Russian Academy of Sciences

Email: albantovaaa@mail.ru
Russian Federation, Moscow

A. N. Goloshchapov

Emanuel Institute of Biochemical Physics Russian Academy of Sciences

Email: albantovaaa@mail.ru
Russian Federation, Moscow

M. A. Korovin

Emanuel Institute of Biochemical Physics Russian Academy of Sciences

Email: albantovaaa@mail.ru
Russian Federation, Moscow

V. V. Kuvyrkova

Emanuel Institute of Biochemical Physics Russian Academy of Sciences

Email: albantovaaa@mail.ru
Russian Federation, Moscow

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2. Fig. 1. FAD fluorescence in mouse splenocytes: single (left) and multiple fluorescence (right) in cells during apoptosis.

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3. Fig. 2. Micrographs of splenocytes during apoptosis induced by sodium anfen (10-4 M) and a 6-fold magnified image of the cell (granules of NAD(P)H-oxidase enzyme complexes NOX are visible on the membrane). Confocal microscope with 100x magnification (fluorophore – Annexin V-FITC).

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4. Fig. 3. Diagram of the content of apoptotic cells in the splenocyte culture (fluorophore – AnnexinV-FITC) – 1, the number of non-viable cells (fluorophore EtBr) – 2. Changes in the cell content under the influence of hydrogen peroxide (5 μM), sodium anfen (10-4 M), and their combined effect.

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5. Fig. 4. Percentage of cells with FAD-1 fluorescence and immunofluorescence of granules of NAD(P)H-oxidase complexes-2 in a splenocyte culture under the influence of hydrogen peroxide (5 μM), sodium anfen (10-4 M) and their combined effect.

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6. Fig. 5. Micrographs of a neutrophil in a splenocyte culture, which is in the stage of NETosis – the release of a DNA network surrounded by NAD(P)H-oxidase complexes – (fluorophore – AnnexinV-FITC (a) and 7AAD (b)). Confocal microscope with 100x magnification.

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