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US 8,465,988 B2Granted
Cell-based antioxidant protection assay
AssigneeNatural Immune Systems, Inc.
InventorGitte S. Jensen
Life of this patent
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Abstract
Methods are provided herein for determining antioxidant activity of a test sample in intact cells. The method includes determining the antioxidant capacity of a test sample in intact red blood cells, wherein the test sample is added to intact red blood cells and oxidative damage is measured by alteration of fluorescence intensity of an oxidation-sensitive fluorescent indicator dye.
Claims22
A method for determining antioxidant activity of a sample comprising:
- adding a sample to intact red blood cells under conditions which permit the sample to cross the cell membrane of the intact red blood cells;
- washing the red blood cells to remove sample which has not crossed the cell membrane of the red blood cells;
- lysing the washed red blood cells;
- adding a fluorescent indicator dye to the lysed red blood cells, wherein the fluorescence indicator dye exhibits oxidation-sensitive changes in fluorescence intensity;
- inducing oxidative stress in the lysed red blood cell mixture;
- and
- detecting fluorescence intensity in the lysed red blood cell mixture using a microplate reader,
- wherein a decrease in the fluorescence intensity of the fluorescence indicator dye in the lysed red blood cells in the presence of the sample as compared to a control comprising lysed red blood cells which have not been treated with the sample or lysed red blood cells treated with a standard with known antioxidant activity indicates the antioxidant activity of the sample.
5 more elements
A method for determining antioxidant activity of a sample comprising:
- adding a sample to intact red blood cells under conditions which permit the sample to cross the cell membrane of the intact red blood cells;
- washing the red blood cells to remove sample which has not crossed the cell membrane of the red blood cells;
- lysing the washed red blood cells, thereby generating lysed red blood cells;
- inducing oxidative stress in the lysed red blood cells;
- and
- detecting fluorescence intensity of hemoglobin in the lysed red blood cells using a microplate reader, wherein hemoglobin exhibits oxidation-sensitive changes in fluorescence intensity,
- wherein a decrease in the fluorescence intensity of the hemoglobin in the lysed red blood cells treated with the sample as compared to a control comprising lysed red blood cells which have not been treated with the sample or lysed red blood cells treated with a standard with known antioxidant activity indicates the antioxidant activity of the sample.
4 more elements
A method for determining antioxidant activity of a sample comprising:
- adding a sample to intact red blood cells under conditions which permit the sample to cross the cell membrane of the intact red blood cells;
- washing the red blood cells to remove sample which has not crossed the cell membrane of the red blood cells;
- lysing the washed red blood cells in water;
- adding 5-(and-6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester to the lysed red blood cells, wherein the 5-(and-6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester exhibits oxidation-sensitive changes in fluorescence intensity;
- adding hydrogen peroxide to the lysed red blood cell mixture;
- and
- detecting fluorescence intensity of the 5-(and-6-)-chloromethyl-2′,7′dichlorodihydrofluorescein diacetate acetyl ester in the lysed red blood cell mixture using a microplate reader, wherein a decrease in the fluorescence intensity in the lysed red blood cells treated with the sample as compared to a control comprising lysed red blood cells which have not been treated with the sample or lysed red blood cells treated with a standard with known antioxidant activity indicates the antioxidant activity of the sample.
4 more elements
Dependencies are inferred from the claim text.