Target cells with specific antigens (e.g., normal cells, tumor cells, virus-infected cells) bind to the corresponding antibody and, with the participation of complement, cause damage to the target cell membrane, leading to increased permeability of the cell membrane and cell death. (Source: Immunology Internship Guidance - Department of Immunology, School of Basic Medical Sciences, Peking University School of Medicine)
Operation method
Complement-mediated cytotoxicity assay
Principle
Target cells with specific antigens (e.g., normal cells, tumor cells, virus-infected cells) bind to the corresponding antibody and, with the participation of complement, cause damage to the target cell membrane, leading to an increase in the permeability of the cell membrane and cell death. Dyes (e.g., eosin-Y, tepan blue) can pass through the cell membrane and enter the cell to color the cell, so they can be used to indicate dead or dying cells, while living cells are not colored. This is the complement-dependent cytotoxicity test, which can be used to check for cell membrane antigens and to identify the specificity of antibodies. In this experiment, Thy-1 antigen is the specific surface antigen of mouse thymus T cells, and the monoclonal antibody against mouse Thy-1 can kill more than 95% of thymocytes in vitro through the synergistic effect of complement.
Materials and Instruments
Mouse Move 1. Preparation of mouse thymus cell suspension Caveat 1. Thymocytes should be prepared quickly and need to be manipulated in an ice bath to maintain cell viability. 2. the potency of anti-Thy-1 monoclonal antibody and complement should be determined in the pre-test. 3. If the number of dead cells in the cell control tube exceeds 5%, the experiment should be redone. 4. False-positive reactions can also result from prolonged periods of non-detection of cells after addition to slides. Common Problems I. Experimental results For more product details, please visit Aladdin Scientific website.
Hank's solution Anti-Mouse Thy-1 Monoclonal Antibody Complement Eosin-Y staining solution
Dissecting instruments Petri dishes Stainless steel mesh Test tubes Pipettes Tip pipettes Slides Coverslips
4-6 weeks old mice were killed by cervical dislocation method, and the thymus gland was removed and put into a petri dish with about 4 ml of cold Hank's liquid. The thymus was removed and placed in a dish with about 4 ml of cold Hank's solution, ground on a 100-mesh stainless steel mesh, sieved, placed in a test tube, and centrifuged at 1000 rpm for 5 minutes. The cells were centrifuged at 1000 rpm for 5 minutes and washed twice with Hank's solution. The precipitated cells were resuspended in Hank's solution to make 1×107 /ml cell suspension. 2. Take 3 test tubes, and then wash them with Hank's solution for 5 minutes at 1000 rpm for 5 minutes.
2. Take 3 test tubes, label the order, and add 1×107 /ml thymus cell suspension, anti-mouse Thy-1 monoclonal antibody and anti-thymus cell suspension according to the following table. Add 1×107/ml thymus cell suspension, anti-mouse Thy-1 monoclonal antibody (optimal dilution) and Hank's solution according to the table below, and place in a 37 ℃ water bath for 30 minutes. 3.
3. After removal, add 1 drop of 1% Eosin-Y dye to each tube, mix well, and leave at room temperature for 2 minutes. 4.
4. After remixing, put a drop of the solution on a slide and cover the slide for microscopic examination. Observe the slides first under low magnification, then under high magnification. Observe first under low magnification and then under high magnification to compare the cell viability in the 3 tubes.
The formula is as follows: