Flow cytometric, whole blood dendritic cell immune function assay
First Claim
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1. A method of measuring dendritic cell function wherein each step in said method is sequentially performed on one whole blood sample, said method comprising:
- (a) contacting a sample of whole blood with a dendritic cell activator and a secretion inhibitor;
(b) contacting said sample with a plurality of dendritic cell distinguishing antibodies and at least one dendritic cell subsetting antibody;
(c) permeabilizing nucleated cells in said sample with a permeabilizing agent;
(d) contacting said sample with at least one cytokine-specific antibody, said cytokine-specific antibody binding to cytokine inside the cell resulting in intracellular binding; and
then (e) flow cytometrically measuring said intracellular binding of said at least one cytokine-specific antibody by dendritic cells that concurrently bind said dendritic cell subsetting antibody, the level of intracellular binding providing a measure of dendritic cell function.
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Abstract
The invention provides a flow cytometric method for measuring dendritic cell function in whole blood, comprising the steps of: (a) contacting a whole blood sample with a dendritic cell activator; (b) adding to the sample a plurality of dendritic cell-distinguishing antibodies and at least one cytokine-specific antibody; and then (c) flow cytometrically assaying the sample for the binding of the cytokine-specific antibody by at least one distinguishable DC subset.
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Citations
24 Claims
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1. A method of measuring dendritic cell function wherein each step in said method is sequentially performed on one whole blood sample, said method comprising:
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(a) contacting a sample of whole blood with a dendritic cell activator and a secretion inhibitor;
(b) contacting said sample with a plurality of dendritic cell distinguishing antibodies and at least one dendritic cell subsetting antibody;
(c) permeabilizing nucleated cells in said sample with a permeabilizing agent;
(d) contacting said sample with at least one cytokine-specific antibody, said cytokine-specific antibody binding to cytokine inside the cell resulting in intracellular binding; and
then(e) flow cytometrically measuring said intracellular binding of said at least one cytokine-specific antibody by dendritic cells that concurrently bind said dendritic cell subsetting antibody, the level of intracellular binding providing a measure of dendritic cell function. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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22. A method of measuring dendritic cell function, comprising:
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(a) contacting a sample of whole blood with a dendritic cell activator;
(b) contacting said sample with a plurality of dendritic cell distinguishing antibodies, at least one dendritic cell subsetting antibody, and at least one antibody specific for a cytokine; and
then(c) flow cytometrically measuring the binding of said at least one cytokine specific antibody that concurrently bind said dendritic cell subsetting antibody, the level of binding of the antibody specific for the cytokine providing a measure of dendritic cell function. - View Dependent Claims (23)
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24. A method of distinguishing dendritic cell subsets wherein each step in said method is sequentially performed on one whole blood sample, said method comprising:
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(a) contacting a sample of whole blood with a dendritic cell activator and a secretion inhibitor;
(b) contacting said sample with a plurality of dendritic cell distinguishing antibodies;
(c) permeabilizing nucleated cells in said sample with a permeabilizing agent;
(d) contacting said sample with at least one cytokine-specific antibody, said cytokine-specific antibody binding to cytokine inside the cell resulting in intracellular binding; and
then(e) flow cytometrically measuring said intracellular binding of said at least one cytokine-specific antibody by dendritic cells, wherein said dendritic cell subsets are distinguished by differences in the amount of said intracellular binding.
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Specification