RU2842069C1 - Method for assessing effect of substances on intensity of radiation-induced oxidative stress on adsorption cell cultures using plate reader - Google Patents

Abstract

FIELD: measuring.

SUBSTANCE: invention relates to examination of biological materials by optical means and to a method for assessing the effect of substances on the intensity of radiation-induced oxidative stress on cell cultures using a plate reader. Method is implemented by introducing a cell suspension in a nutrient medium into the wells of the plate with subsequent incubation, after which the solutions of the analysed substances are added into the samples with subsequent incubation. Samples are exposed to radiation, after which fluorescent dyes dichlorodihydrofluorescein and Hoechst or DAPI are added to the samples. Further, the cells are incubated with subsequent complete or partial replacement of the medium above the cells, after which fluorescence of the dyes with which the cells were stained is recorded. Oxidative stress intensity is assessed by the relation of fluorescence intensity of the oxidised form of dichlorodihydrofluorescein to fluorescence intensity of Hoechst or DAPI dye.

EFFECT: possibility of obtaining objective numerical data on the intensity of oxidative stress processes in exposed cells under the effect of various substances with possibility of further statistical processing.

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Description

The invention relates to the field of biophysics, molecular biology, medicine and veterinary science and can find application in studying oxidative stress processes in models of adsorption cell cultures.

The state of oxidative stress as an increase in the content of free radicals and other active forms of oxygen is one of the main components of the biological action of radiation of various natures. These include ionizing radiation of electromagnetic nature, such as gamma radiation and X-rays, and corpuscular radiation, such as neutron, proton, beta radiation, accelerated ions, and non-ionizing radiation, both of electromagnetic nature, and acoustic radiation, which are mechanical vibrations of various frequencies.

The search for means capable of mitigating the negative effects of radiation on the body is very relevant; and an important role here is given to substances that reduce the severity of oxidative stress. Substances, the use of which leads to an increase in the severity of radiation-induced oxidative stress, in turn, can be useful in radiation therapy and brachytherapy of oncological diseases.

At the same time, it is extremely important to develop methods that allow easy and rapid assessment of the antioxidant properties of substances on biological models. And the use of cell cultures for these purposes seems justified.

In experiments on cell cultures, various fluorimetric research methods are often used [Ozolina N.V., Nurminsky V.N., Rakevich A.L., Kolesnikova E.V., Nesterkina I.S., Donskaya L.I., Salyaev R.K. The effect of oxidative stress on the vacuolar membrane // Biological membranes. - 2014. - Vol. 31, No. 4. P: 263-269. DOI: 10.7868/S0233475514040070; Morosanova M.A., Plotnikov E.Yu., Pevzner I.B., Zorova L.D., Zorov D.B. Kidney cell death during inflammation: the role of oxidative stress and mitochondria // Biological membranes. - 2013. - Vol. 30, No. 5-6. S: 445-453. DOI: 10.7868/S0233475513050113; Saleem M, Ahmad T, Haynes A.R., Albritton C.F., Mwesigwa N., Graber M.K., Kirabo A., Shibao C.A. Innovative assessment of lipid-induced oxidative stress and inflammation in harvested human endothelial cells // Physiological Reports. - 2024. - Vol. 12(11). P. e16048. DOI: 10.14814/phy2.16048].

The potential effectiveness of various radioprotective drugs, and vice versa - radiosensitizers, can be assessed by their effect on the severity of oxidative stress in cell culture. This can be done by recording the fluorescence intensity of dyes sensitive to active forms of oxygen. Such a fluorochrome is dichlorofluorescein (DCF) and its various derivatives, such as dichlorofluorescein diacetate, carboxydichlorofluorescein, etc. To be able to work with a large number of samples, it is advisable to use plate readers.

To obtain adequate results, it is necessary to use a suitable experimental design. In particular, it is necessary that the fluorescent signal is registered only from the probe located inside the cells: a fluorophore glowing in the culture medium will lead to a false interpretation of the study results in the direction of an allegedly high level of oxidative stress. To avoid this effect, it is necessary to wash off the fluorophore located outside the cells after staining the samples. In the case of an adsorption culture, washing is carried out by replacing the culture medium above the cells.

And since different samples may have different numbers of cells, a situation may arise where a sample has few cells, but the oxidative stress in them is significant. And in another sample, the content of active oxygen forms is low, so the DCF fluorescence from one cell is insignificant, but due to the large number of cells in the sample, the total DCF fluorescence intensity may even exceed that from a sample with a small number of cells experiencing pronounced oxidative stress.

Therefore, it is necessary to normalize the fluorescence intensity of the fluorophore detecting active forms of oxygen to the number of cells in the sample. Since counting cells in a sample using a Goryaev chamber or even automatic cell counters takes a lot of time, and, in addition, to perform this counting it is necessary to disrupt the attachment of cells to the substrate, it seems advisable to use a fluorescent dye that glows when bound to DNA. Since the DNA content in one cell is, on average, the same, the fluorescence intensity of this dye will be proportional to the number of cells in the sample.

Therefore, if the fluorescence intensity of DCF or its derivative is divided by the fluorescence intensity of a dye specific for DNA molecules, it is possible to obtain a value characterizing the severity of oxidative stress in one average cell in the studied sample. DAPI or Hoechst can be used as fluorescent dyes to characterize the number of cells in the sample.

The essence of the proposed method is to assess the severity of oxidative stress in irradiated cells when exposed to various substances by staining the cells with fluorescent dyes such as dichlorodihydrofluorescein or its derivatives, glowing when oxidized by active oxygen species, and dyes glowing when bound to DNA, such as Hoechst or DAPI. The fluorescence intensity of the oxidized form of dichlorodihydrofluorescein - dichlorofluorescein (DCF) - is higher, the higher the content of active oxygen species (ROS) in the cell. And by dividing this value by the fluorescence intensity of the dye that binds to DNA and glows brighter, the more cells containing DNA in the sample, it becomes possible to obtain a value characterizing the average value of the severity of oxidative stress processes in a specific cell in the sample under study.

The closest to the proposed method is the method [Almammadov T., Elmazoglu Z., Alkan G.A., Kepil D., Aykent G., Kolemen S., Gunbas G. Locked and Loaded: P-Galactosidase Activated Photodynamic Therapy Agent Enables Selective Imaging and Targeted Treatment of Glioblastoma Multiforme Cancer Cells // ACS Applied Bio Materials. - 2022. - Vol. 5(9), P.: 4284-1293. DOI:10.1021/acsabm.2c00484], which involves assessing the intensity of oxidative stress processes in glioblastoma cells by microscopy using cell staining with Hoechst and DCF dyes. Staining of cells with these two dyes allowed the assessment of cells in a state of oxidative stress (ROS induced DCF fluorescence) in micrographs (due to Hoechst staining, which binds to DNA).

The disadvantage of the prototype method is the need for rather labor-intensive preparation of micropreparations and large time expenditures required to implement the method. In addition, visual assessment of fluorescence brightness implies a high degree of subjectivity in assessing the severity of oxidative stress processes in cells, which almost completely excludes statistical processing of the results.

The proposed method involves the use of a tablet reader, which implies simultaneous work with several dozen samples. In this case, there is no need to prepare a micropreparation, which requires additional time, and it is possible to study changes in the severity of oxidative stress over time, and the obtained numerical results can easily be subjected to statistical processing.

The proposed method is aimed at solving the technical problem of the actual impossibility of obtaining objective numerical information suitable for further statistical processing on the severity of oxidative stress processes in irradiated cells when exposed to various substances and the problem of high labor intensity and time costs in preparing micropreparations and visual analysis of information, without which it is impossible to use the microscopy method described in the prototype method.

The technical result is the possibility of obtaining objective numerical data on the severity of oxidative stress processes in irradiated cells under the influence of various substances with the possibility of further statistical processing, including due to the possibility of conducting fluorescence measurements from a large number of samples, which is impossible when using the fluorescence microscopy method, but is possible when conducting the corresponding study using a plate reader.

The method for assessing the effect of substances on the severity of oxidative stress induced by radiation on adsorption cell cultures using a plate reader is implemented by introducing a cell suspension in a nutrient medium into the wells of the plate, followed by incubation for 6-72 hours, followed by adding solutions of the substances being studied to the samples, followed by incubation for 0.5-48 hours, after which the samples are exposed to ionizing, non-ionizing or acoustic radiation, after which the fluorescent dyes dichlorodihydrofluorescein or its derivative and Hoechst or DAPI are introduced into the samples, in the presence of which the cells are incubated, followed by a complete or partial replacement of the medium above the cells, after which either the fluorescence of the dyes with which the cells were stained is recorded, or the substances being studied are additionally introduced, followed by incubation, after which the fluorescence of the dyes with which the cells were stained is recorded, and the severity of oxidative stress is assessed by the intensity ratio fluorescence intensity of the oxidized form of dichlorodihydrofluorescein or its derivative to the fluorescence intensity of Hoechst or DAPI dye.

The proposed method will allow to study the effect of various substances on the processes of oxidative stress in cells caused by the effect of radiation of various nature on these cells with the least labor costs and with a high degree of objectivity of the obtained data. This will be achieved by using automatic systems for recording plate readers of fluorescence intensity instead of visual assessment inherent in the microscopy method. The magnitude of oxidative stress in a separate cell in the studied sample is characterized by the ratio of the fluorescence intensity of DCF or its derivative to the fluorescence intensity of Hoechst or DAPI dyes.

The proposed method for assessing the effect of substances on oxidative stress caused by radiation by initial incubation of cells in the absence of the substances under study allows one to neutralize possible inhibition of the rate of cell attachment to the substrate and the rate of monolayer formation under the action of these substances. Further incubation of cells in a medium containing the substances under study will allow one to study their effect on cells. In this case, this effect may be due to both a change in cell metabolism due to the effect of substances during incubation prior to irradiation and the interaction of these substances with free radicals and active oxygen species formed during irradiation.

The possible influence of the studied substances on the recovery of cells after irradiation can be studied by incubating cells with the studied substances after washing the samples from fluorescent dyes that did not penetrate the cells.

Examples of using the method

Example No. 1. Evaluation of the effect of a mixture of ascorbic acid and copper chlorophyllin on the severity of oxidative stress caused by exposure to neutron radiation on AN-130 rat liver hepatoma cells

Add a suspension of AN-130 rat liver hepatoma cells to the wells of the plate and incubate the samples for 24 hours in a CO ₂ incubator at 37°C. Then add copper chlorophyllin and ascorbic acid solutions to the samples and incubate the samples at 37°C for 3 hours. Then expose the samples to neutron radiation. After irradiation, add dichlorodihydrofluorescein solution to the samples and incubate the samples for 20 minutes, then add DAPI dye to the samples and incubate the samples for 10 minutes. Next, remove the medium above the cells and replace it with a mixture of copper chlorophyllin and ascorbic acid. Incubate the samples for two hours at 37°C. Next, replace the medium above the cells with phosphate buffer and measure DCF and DAPI fluorescence in the samples using a plate reader. The severity of oxidative stress in samples is assessed as the ratio of DCF fluorescence intensity to DAPI.

Example No. 2. Evaluation of the effect of succinic acid on the severity of oxidative stress caused by exposure to X-ray radiation on human lung adenocarcinoma A549 cells.

Add a suspension of human lung adenocarcinoma A549 cells to the wells of the plate and incubate the samples for 12 hours in a CO ₂ incubator at 37°C. Then add succinic acid solutions of the studied concentrations to the samples and incubate the samples at 37°C for 2 hours. Then expose the samples to X-ray radiation. After irradiation, add dichlorodihydrofluorescein solution to the samples and incubate the samples for 20 minutes, then add Hoechst-33342 dye to the samples and incubate the samples for 10 minutes. Then remove the medium above the cells and add succinic acid solutions instead. Incubate the samples for one hour at 37°C. Next, replace the medium above the cells with phosphate buffer and measure the fluorescence of DCF and Hoechst-33342 in the samples using a plate reader. The severity of oxidative stress in samples is assessed as the ratio of the fluorescence intensity of DCF to Hoechst-33342.

Example No. 3. Evaluation of the effect of a mixture of indralin and tartaric acid on the severity of oxidative stress caused by exposure to alpha radiation on rat bladder carcinoma HB-CLS-3 cells.

Add a suspension of HB-CLS-3 rat bladder carcinoma cells to the wells of the plate and incubate the samples for 36 hours in a CO ₂ incubator at 37°C. Next, add a mixture of indralin and tartaric acid to the samples and incubate the samples at 37°C for 12 hours. Next, expose the samples to alpha radiation by adding alpha-emitting radionuclides to the samples. After replacing the medium above the cells, add a solution of carboxydichlorodihydrofluorescein to the samples and incubate the samples for 20 minutes, then add Hoechst-33342 dye to the samples and incubate the samples for 10 minutes. Next, remove the medium above the cells and replace it with pure culture medium. Then incubate the samples for 30 minutes at 37°C. Next, it is necessary to replace the medium above the cells with phosphate buffer and measure the fluorescence of DCF and Hoechst-33342 in the samples using a plate reader. The severity of oxidative stress in the samples is estimated as the ratio of the fluorescence intensity of DCF to Hoechst-33342.

Example No. 4. Evaluation of the effect of amygdalin on the severity of oxidative stress caused by exposure to microwave radiation on a rabbit fibroblast cell line.

Add a suspension of rabbit fibroblasts to the wells of the plate and incubate the samples for 72 hours in a CO ₂ incubator at 37°C. Then add amygdalin solutions of the studied concentrations to the samples and incubate the samples at 37°C for 4 hours. Then expose the samples to microwave radiation. After irradiation, add a solution of dichlorodihydrofluorescein to the samples and incubate the samples for 20 minutes, then add Hoechst-33342 dye to the samples and incubate the samples for 10 minutes. Then remove the medium above the cells and add amygdalin solutions instead. Incubate the samples for one hour at 37°C. Next, replace the medium above the cells with phosphate buffer and measure the fluorescence of DCF and Hoechst-33342 in the samples using a plate reader. The severity of oxidative stress in samples is assessed as the ratio of the fluorescence intensity of DCF to Hoechst-33342.

Example No. 5. Evaluation of the effect of copper chlorophyllin on the severity of oxidative stress when exposed to visible light on human skin epidermoid carcinoma A-431 cells.

Add a suspension of human epidermoid skin carcinoma A-431 cells to the wells of the plate and incubate the samples for 12 hours in a CO ₂ incubator at 37°C. Then add copper chlorophyllin solutions to the samples and incubate the samples at 37°C for an hour. Then expose the samples to light in the visible range. After irradiation, add dichlorodihydrofluorescein solution to the samples and incubate the samples for 20 minutes, then add DAPI dye to the samples and incubate the samples for 10 minutes. Then remove the medium above the cells and replace it with clean medium. Incubate the samples for two hours at 37°C. Next, replace the medium above the cells with phosphate buffer and measure DCF and DAPI fluorescence in the samples using a plate reader. The severity of oxidative stress in samples is assessed as the ratio of DCF fluorescence intensity to DAPI.

Example No. 6. Evaluation of the effect of potassium malate on the intensity of free-radical reactions when exposed to continuous ultrasound on cat bone marrow stromal cells.

Add cat bone marrow stromal cell suspension to wells of plate and incubate samples for 48 hours in CO ₂ -incubator at 37°C. Then add potassium malate solutions of the studied concentrations to samples and incubate samples at 37°C for 12 hours. Then expose samples to ultrasound in continuous mode. After sonication, add dichlorodihydrofluorescein solution to samples and incubate samples for 20 minutes, then add Hoechst-33342 dye to samples and incubate samples for 10 minutes. Then remove the medium above the cells and add potassium malate solutions instead. Incubate samples for 3 hours at 37°C. Then it is necessary to replace the medium above the cells with phosphate buffer and measure DCF and Hoechst-33342 fluorescence in samples using a plate reader. The severity of oxidative stress in samples is assessed as the ratio of the fluorescence intensity of DCF to Hoechst-33342.

Example No. 7. Evaluation of the effect of sodium succinate on the intensity of free-radical reactions when exposed to ultrasound in pulsed mode on stromal cells of dog bone marrow.

Add a suspension of canine bone marrow stromal cells to the wells of the plate and incubate the samples for 48 hours in a CO ₂ incubator at 37°C. Then add sodium succinate solutions of the studied concentrations to the samples and incubate the samples at 37°C for 12 hours. Then expose the samples to ultrasound in pulsed mode. After sonication, add a solution of dichlorodihydrofluorescein to the samples and incubate the samples for 20 minutes, then add DAPI dye to the samples and incubate the samples for 10 minutes. Then remove the medium above the cells and add sodium succinate solutions instead. Incubate the samples for 3 hours at 37°C. Next, replace the medium above the cells with phosphate buffer and measure the fluorescence of DCF and DAPI in the samples using a plate reader. The severity of oxidative stress in samples is assessed as the ratio of DCF fluorescence intensity to DAPI.

Example No. 8. Evaluation of the effect of a mixture of riboxin and sodium ascorbate on the intensity of free-radical reactions when a culture of goat fibroblasts is exposed to infrasound.

Add a suspension of goat fibroblasts to the wells of the plate and incubate the samples for 36 hours in a CO ₂ incubator at 37°C. Then add solutions of riboxin and sodium ascorbate of the studied concentrations to the samples and incubate the samples at 37°C for 24 hours. Then expose the samples to infrasound in continuous mode. After sonication, add a solution of dichlorodihydrofluorescein to the samples and incubate the samples for 20 minutes, then add Hoechst-33342 dye to the samples and incubate the samples for 10 minutes. Then remove the medium above the cells and add solutions of riboxin and sodium ascorbate instead. Incubate the samples for 4 hours at 37°C. Next, it is necessary to replace the medium above the cells with phosphate buffer and measure the fluorescence of DCF and Hoechst-33342 in the samples using a plate reader. The severity of oxidative stress in the samples is estimated as the ratio of the fluorescence intensity of DCF to Hoechst-33342.

Example No. 9. Evaluation of the effect of a mixture of riboxin and bovine placenta hydrolysate on the intensity of free-radical reactions when exposed to ultraviolet radiation on a mouse fibroblast culture.

Add a suspension of mouse fibroblasts to the wells of the plate and incubate the samples for 36 hours in a CO ₂ incubator at 37°C. Then add a mixture of riboxin and bovine placenta hydrolysate to the samples and incubate the samples at 37°C for 24 hours. Then expose the samples to ultraviolet radiation. After irradiation, add a solution of dichlorodihydrofluorescein to the samples and incubate the samples for 20 minutes, then add Hoechst-33342 dye to the samples and incubate the samples for 10 minutes. Then remove the medium above the cells and replace it with a mixture of riboxin and bovine placenta hydrolysate. Incubate the samples for 2 hours at 37°C. Next, it is necessary to replace the medium above the cells with phosphate buffer and measure the fluorescence of DCF and Hoechst-33342 in the samples using a plate reader. The severity of oxidative stress in the samples is estimated as the ratio of the fluorescence intensity of DCF to Hoechst-33342.

Example No. 10. Evaluation of the effect of a mixture of cytochrome C, tetraoleoylcardiodipine and tocopherol on the intensity of free-radical reactions when exposed to gamma radiation on a DSL-6A-C1 culture of rat pancreatic duct carcinoma.

Add a suspension of rat pancreatic ductal carcinoma DSL-6A-C1 cells to the wells of the plate and incubate the samples for 12 hours in a CO ₂ incubator at 37°C. Then add solutions of cytochrome C, tetraoleoylcardiodipine and tocopherol to the samples and incubate the samples at 37°C for 2 hours. Then expose the samples to gamma radiation. After irradiation, add a solution of dichlorodihydrofluorescein to the samples and incubate the samples for 20 minutes, then add DAPI dye to the samples and incubate the samples for 10 minutes. Then remove the medium above the cells and replace it with clean medium. Incubate the samples for two hours at 37°C. Then replace the medium above the cells with phosphate buffer and measure the fluorescence of DCF and DAPI in the samples using a plate reader. The severity of oxidative stress in the samples is assessed as the ratio of the fluorescence intensity of DCF to DAPI.

Claims (1)

A method for assessing the effect of substances on the severity of oxidative stress induced by radiation on adsorption cell cultures using a plate reader, which is implemented by introducing a cell suspension in a nutrient medium into the wells of a plate, followed by incubation for 6-72 hours, followed by adding solutions of the substances being studied to the samples, followed by incubation for 0.5-48 hours, after which the samples are exposed to ionizing, non-ionizing or acoustic radiation, after which the fluorescent dyes dichlorodihydrofluorescein or its derivative and Hoechst or DAPI are added to the samples, in the presence of which the cells are incubated, followed by a complete or partial replacement of the medium above the cells, after which either the fluorescence of the dyes with which the cells were stained is recorded, or the substances being studied are additionally added, followed by incubation, after which the fluorescence of the dyes with which the cells were stained is recorded, and the severity of oxidative stress is assessed by the ratio the fluorescence intensity of the oxidized form of dichlorodihydrofluorescein or its derivative to the fluorescence intensity of Hoechst or DAPI dye.