1, the principle of experiment Target cells with specific antigens (such as normal cells, tumor cells, and virus-infected cells) bind to the corresponding antibodies, and in the presence of complement, cause damage to the target cell membrane, resulting in increased permeability of the cell membrane and cell death. Dyes (eg, eosin-Y, trypan blue) can enter the cells through the cell membrane to stain the cells, so they can be used to indicate dead cells or dying cells, while living cells are not colored. This is a complement-dependent cytotoxicity assay, which can be used to examine cell membrane antigens and to identify antibody specificity. In this experiment, Thy-1 antigen is a mouse thymic T cell-specific surface antigen, which can kill more than 95% of thymocytes by the synergistic action of complement in vitro using a monoclonal antibody against mouse Thy-1. 2, experimental materials (1) Anatomical instruments (ophthalmic scissors, ophthalmology), plates, 80-100 mesh stainless steel mesh; (2) test tube, lml pipette, tip pipette; (3) slides and coverslips; (4) C57BL/6J mice; (5) Cold Hank's solution containing 5% NBS; (6) monoclonal antibody against mouse Thy-1 (optimal dilution, prepared in our laboratory); (7) Complement (guinea pig fresh serum and absorbed by mouse thymocytes, pre-determined titer and diluted to the optimal dilution); (8) 1% Yihong-Y dye solution. 3. Experimental methods (1) Preparation of mouse thymocyte suspension: 4 to 6 weeks old mice were sacrificed by cervical dislocation, and the thymus was taken out and placed in a dish containing about 4 ml of cold Hank's solution, and ground on a 100-mesh stainless steel mesh. The sieve was placed in a test tube, centrifuged at 1000 rpm for 5 minutes, and washed twice with Hank's solution. The precipitated cells were resuspended in Hank's solution to prepare a 1 x 107/ml cell suspension. (2) Take 3 tubes, indicate the order, and add 1×107/ml thymocyte suspension, anti-mouse Thy-1 monoclonal antibody (optimal dilution) and Hank's solution according to the following table, and put in 37°C. Water bath for 30 minutes. (3) After taking out, add 1 drop of 1% eosin-Y dye solution to each tube, mix and let stand for 2 minutes at room temperature. (4) After remixing, drop the tablets on a slide and add a cover glass to the microscope. First observe under low magnification, then observe with high power microscope, compare the cell life and death in 3 tubes. 4. Experimental results The dead cells are red, dull and swollen; the living cells are not colored, shiny and normal. 200 cells were counted under high magnification and the percentage of dead cells was counted. The calculation formula is as follows: Percentage of dead cells = EQ EQ \F (% of dead cells in the experimental tube - % of dead cells in the control tube, 100% - % of dead cells in the control tube) 5, matters needing attention (1) The preparation of thymocytes is fast and needs to be operated in an ice bath to maintain cell viability; (2) The titer of anti-Thy-1 monoclonal antibody and complement should be determined in the preliminary experiment; (3) If the number of dead cells in the cell control tube exceeds 5%, the experiment needs to be done again; (4) A long-term placement of cells after being added to a glass slide without detection may also result in a false positive reaction. Frozen Seafood Mix,Mixed Seafood Bags,Frozen Seafood Mix Bags,Frozen Seafood Cocktail Zhoushan Haiwang Seafood Co., Ltd. , https://www.haiwangseafoods.com Experimental Materials Test tube Complement control tube Cell control tube 1×10 7 /ml thymocyte suspension 0.1ml 0.1ml 0.1ml Thy-1 monoclonal antibody 0.1ml —— —— Hank's solution —— 0.1ml 0.2ml 1:3 complement 0.1ml 0.1ml ——