Method for improving detection rate of cytokines secreted by peripheral blood leukocytes

Abstract

The invention discloses a method for improving the detection rate of cytokines secreted by peripheral blood leukocytes. The method comprises the following steps: S1, taking whole blood and adding a culture solution to dilute the whole blood; S2, adding phorbol ester and ionomycin calcium salt, carrying out culturing for 6 hours, and adding monensin in the later 4 hours of the culture so as to obtain cells to be detected; S3, adding a cell surface labeled antibody into the cells to be detected for staining of the cells to be detected, dissolving red blood cells with a whole blood sample, carrying out washing with a staining buffer, adding polyformaldehyde for lucifugal immobilization, adding a membrane permeating buffer for membrane permeation, adding bovine serum albumin and mouse serum for blocking, adding a cytokine antibody for staining, carrying out washing with membrane permeating buffer, and carrying out washing with a staining buffer to obtain stained cells; and S4, carrying outdetection with a flow cytometer. The method of the invention has a wide application range and high ratio of cytokine secreting cells, and experimental data and control data have statistical significance.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for improving the detection rate of cytokines secreted by peripheral blood leukocytes. Background technique [0002] Flow cytometry is a common method used by most researchers to detect secreted cytokines in peripheral blood samples. In experiments, it is customary to separate mononuclear cells (PBMC) in peripheral blood samples or directly sort T cell subsets for detection. However, for some cytokines with relatively small secretion, such as IL-17, IL-4, IL-22, etc., because the final data obtained are relatively low, many experimental data are not statistically significant when compared with the control group. Because the secretion of one cytokine requires the assistance of other cytokines, and there are many cytokines in serum, as reported by Manel N et al., in the absence of serum, initial CD4+ T cells secrete IL-17 dependent on TGF- Presence of β, IL-1β, IL-6,...

AI Technical Summary

Problems solved by technology

However, for some cytokines with relatively small secretion, such as IL-17, IL-4, IL-22, etc., because the final data obtained are relatively low, many experimental data are not statistically significant when compared with the control group

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