T cells usually require two signals to be fully activated. A first signal of antigen specificity is provided through the T-cell receptor (TCR). The TCR interacts with peptide-MHC molecules on the membrane of antigen-presenting cells (APCs). The second signal, the co-stimulatory signal, is non-antigen-specific. It is provided by the interaction between costimulatory molecules expressed on the APC membrane and T cells.
T cell stimulation is required for T cell proliferation, differentiation and survival. Creative Biolabs offers costimulation assays. Specific antibodies are used to mimic first and second signals in in vitro experiments to induce proliferative responses in resting lymphocytes. Below we briefly describe the costimulation protocol to study its modulation of T-cell function and to validate new targets for immunotherapy.
|Stages||Solutions and Reagents|
|Cell Preparation||Phosphate buffer (PBS), lymphocyte isolation solution, culture media|
|Costimulation||Anti-CD3 antibody, anti-CD28 antibody, washing buffer|
Collect target samples and isolate cells using lymphocyte isolation solution. Then collect the target cells and resuspend them in conditioned medium. Finally, count the cells and adjust the concentration of the cell suspension.
Add equal amounts of T cells to each well of the well plate, and set up positive control, negative control, and reference control groups respectively. Then add anti-CD3 antibodies that specifically recognize the surface molecules of T cells and incubate for an appropriate time. Finally, wash away the excess antibody solution.
Add costimulatory molecule-specific antibodies to the corresponding wells as a detection group. Costimulation with anti-CD3 antibody and anti-CD28 antibody is used as a positive control. A single stimulus is used as a reference control. T cells without any stimulation is used as a negative control. All reactions are incubated under consistent and appropriate conditions.
At the end of incubation, stain the cells to be tested with specific fluorescent dyes. Then place them in sample tubes for detection by flow cytometry. The value from the single anti-CD3 stimulus on each plate is taken as 0% costimulation value. And the value from the anti-CD3+ anti-CD28 stimulus is considered as a signal for 100% costimulation. The costimulatory effect to be assessed can be calculated from the standard curve.
Weak or no fluorescence signal
High fluorescent signal
If you encounter any problems, you can briefly describe your experiment in the inquiry form and we will have a professional technical team to answer your questions.
For research use only. Not intended for any clinical use.