The Establishment Of Expanding Gammadelta T Cells In Vitro, The Isolation Of I-IELs And Immunofluorescence Techniques

Purpose:Immunology experimental techniques, which play important roles in keeping the efficiency of experiment, are the base stone of Immunological research work. Thus I put the establishing immunology experimental techniques for my lab as my main work of master phase. In these three years, I totally established three different kinds of immunology experimental techniques, which are expandingγδT cells in vitro, the isolation of i-IELs(small intestine intra-epithelial lymphocytes) from mice and integration of immunohistochemistrical staining into tumor microenvironment research.1. As theγδT cells are one kind of very rare T cells subset in mice peripheral immune system, it is quite hard to get enoughγδT cells to complete the mice transfer model or co-culture experiment. In order to solve this problem, I establish a method to expandγδT cells in vitro, which takes advantage of anti-γδTCR antibody, anti-CD28 antibody and IL-2 to stimulate y8 T cells. ThenγδT cells could expand in vitro.2. AlthoughγδT cells are very rare in peripheral immune system, there are a large amount of them in intra-epithelial of mouse small intestine. However, tough technique issue interferes the normal efficient research work, because it is difficult to isolate high purity i-IELs. In order to overcome this obstacle, I take advantage of two important properties:one is the susceptibility of small intestine intra-epithelial lymphocytes to get off from the basement membrane in hypoxic conditions. The other one is EDTA has the chelation of Ca2+ and Mg2+. So I can isolate i-IELs with physical method. Then these cells could be used in other experiments.3. In order to do the research on the location of Vyl and Vy4γδT cells in tumor microenvironment, I try to integrate the immunohistochemistrical staining into this issue. The most hard points are how to isolate melanoma from skin, how to pretreat the tissue, how to choose the temperature in frozen section and how to choose the optimal fluorescent antibody. By solving this technique issue, now our lab can not only finish the work on the relationship between Vyl T cells and Vy4γδT cells, but also supply an effective tool to research the infiltration of CD4+ T cells, CD8+ T cells or dendritic cells in other kinds of tumors.