Method for identifying gamma delta T cell killing

Abstract

The invention discloses a killing method for identifying gamma delta T cells simply and rapidly. The method comprises the following steps: firstly, healthy volunteer peripheral blood is collected, separation is carried out by utilization of a human lymphocyte separating medium, mononuclear cells are obtained, phosphate antigens HMBPP with 10-30ng / ml and rhIL-2 factors with 500-1000 IU / ml are added, PBMC induction into gamma delta T cells is carried out and amplification culture is carried out; secondly, after culture is carried out for 8-10 days, gamma delta T cells are subjected to detection of destruction experiments, the living cell dye calcein-AM is employed to dye tumor cells, the cell density during dyeing is controlled at 10<5>-10<7> / ml; thirdly, the tumor cells after dyeing and the gamma delta T cells are cultured together for a period of time, the cells are collected, propidium iodide is added for dyeing before detection, finally, a flow cytometer is employed to detect cell death amount, and the cell killing rate is obtained. Through the method that the cell killing efficiency can be detected rapidly through a flow cytometer, limitation conditions of traditional 51Cr radioactive substance pollution and low sensitivity of MTT and LDH are overcome.

Description

technical field [0001] The invention belongs to the technical field of cellular immunology, and specifically uses the principle of flow cytometry to detect and identify the killing efficiency of γδT cells by double-labeling Calcein-AM and PI staining. Background of the invention [0002] T lymphocytes are divided into two types according to the expression of T cell antigen receptor (Tcell receptor, TCR): RCRαβ cells and TCRγδ cells, in which γδT cells are a unique group of T lymphocytes that bridge innate immunity and adaptive immunity, γδT It is mainly distributed in the skin and mucosal tissues, accounting for 0.5-5% of the peripheral lymphocyte population. Such a group of cells plays an important role in the body's anti-infection and anti-tumor immunity. Clinical experiments have confirmed the biological role of γδT cells in tumors. Good therapeutic effect and wide application have been obtained in the treatment, but how to detect the function of the γδT cells expanded an...

AI Technical Summary

Problems solved by technology

[0002] T lymphocytes are divided into two types according to the expression of T cell antigen receptor (Tcell receptor, TCR): RCRαβ cells and TCRγδ cells, in which γδT cells are a unique group of T lymphocytes that bridge innate immunity and adaptive immunity, γδT It is mainly distributed in the skin and mucosal tissues, accounting for 0.5-5% of the peripheral lymphocyte population. Such a group of cells plays an important role in the body's anti-infection and anti-tumor immunity. Clinical experiments have confirmed the biological role of γδT cells in tumors. Good therapeutic effect and wide application have been obtained in the treatment, but how to perform functional detection on the γδT cells expanded and cultured in vitro? The present invention makes a public statement on this item, and the classic detection method of immune cell killing experiment is to use 51 Cr release method, but due to 51 Cr is a radioactive substance, which is inconvenient to operate and other reasons limit the wide application of this method, while MTT method and lactate dehydrogenase (LDH) release method have low sensitivity and other reasons that also limit their wide application. The present invention uses Calcein-AM and PI double-labeled staining combined with flow cytometry to detect immune cell killing efficiency