Antibody-dependent cell-mediated cytotoxicity ( ADCC ) is one of the ways in which antibodies work, when IgG antibodies interact with target cells (virus-infected cells and tumor cells) surface antigenic determinants through the Fab fragment After specific binding, its Fc segment can bind to effector cells such as FcγR killer cells ( NK cells, monocyte-macrophages, neutrophils), trigger the killing activity of effector cells, and directly kill target cells (virus infection Cells and tumor cells).
ADCC is an ideal mechanism for antibody drugs to kill target cells, and the anti-tumor activity of monoclonal antibodies can be verified by ADCC test. Early ADCC verification methods are mostly based on freshly prepared peripheral blood mononuclear cells or NK cells as effector cells. However, these methods have disadvantages such as difficulty in cell classification and culture, cumbersome operation, high background value, and being affected by individual differences. .
iCarTab is committed to the research and development of tumor antibody drugs. In order to solve the common problems in the traditional ADCC test, we used the luciferase reporter gene detection system as the platform to construct stable transfection of CD16 receptor and NFAT (Nuclear Factor of Activated T -cells) Jurkat-NFAT-Luc-CD16 cell line of the original reaction . When an antibody Fab after antigen-binding segment to the target cell, antibody Fc segments effector cells of Jurkat-NFAT-Luciferase- CD16 cell surface (FcyRIIIA) binding, causing -CD16 Jurkat-NFAT-Luciferase (New ) intracellular NFAT associated The signal pathway is activated, which in turn leads to an increase in the expression level of luciferase.
Species: Human peripheral blood leukemia T cells
Storage system: -80°C short-term storage, liquid nitrogen long-term storage
Culture conditions: 1640+20%FBS
Freezing solution: 90% 1640+10% FBS
Growth type: Suspended growth
Transportation method: Fresh cells are transported at room temperature, frozen cells are transported on dry ice
1. Put on a protective mask and antifreeze gloves, remove the cells from the liquid nitrogen tank, and quickly put them in a 37°C water bath to thaw.
2. After the cells are thawed, the cells are delivered to the cell chamber through the delivery chamber.
3. In the biological safety cabinet, take out a 15mL sterile centrifuge tube, and use a 1mL pipette to add 4mL medium to the 15mL centrifuge tube .
4. Use a 75% medical disinfectant alcohol cotton ball to carefully wipe the outer surface of the cell cryopreservation tube thawed in a 37°C water bath for surface disinfection. In the biological safety cabinet, unscrew the cell cryopreservation tube, then use a 1mL pipette to aspirate the cell suspension in the tube, and add it dropwise to the 15mL centrifuge tube containing 4mL medium prepared above , and screw the lid of the 15mL centrifuge tube. Tightly, slowly turn upside down 4 times to mix the cell suspension.
5. Place the centrifuge tube containing the cell suspension in the centrifuge and set the centrifuge parameters as follows: Centrifuge at 200xg at room temperature (~25°C) for 5 minutes.
6. After centrifugation, gently transfer the centrifuge tube to the biological safety cabinet. At this time, white cell clusters can be seen at the bottom of the cytocentrifuge tube. Use an electric aspirator to aspirate the medium supernatant (the cell cluster is relatively loose, be careful not to touch the cell cluster at the bottom).
7. Use a 1mL pipette to add 2mL of complete medium, gently pipette 5 times to resuspend the cell pellet, count, and inoculate.
8. Place the cells in an incubator for overnight culture.