Proper testing of antigen specific T cells and PBMC is essential to ensure antigen specificity and cell functionality. Whether you’re purchasing human cells for research from a supplier or preparing your own antigen specific T cells or PBMC for experimentation, it’s important to perform the appropriate tests beforehand.
At Astarte, we always test our antigen specific T cells and PBMC thoroughly before making them available for purchase. To provide more transparency and insight into how we prepare and test our cells before shipping to you, we’ve described our processes below. If you already have antigen specific T cells or PBMC, you can use these or similar processes to test your cells prior to starting an experiment.
Testing of Antigen Specific T Cells
We demonstrate the antigen specificity of our T cells in two ways:
- Staining with the specific HLA-peptide multimer
- Performing a functional assay, such as proliferation or cytokine production
HLA-peptide multimers are commercially available for some of the most frequently studied antigens and can quickly reveal the proportion of T cells that are specific for that HLA-peptide combination. To stain the cells with HLA-peptide multimer, we recommend following the directions from the supplier of the staining reagent.
Functional assays cannot provide the proportion of antigen specific cells, but are necessary in order to study the effects of drugs and antibodies on T cell activity. Here is our functional testing process for T cells:
- Inactivate antigen-presenting cells. If you measure proliferation, it is essential to measure the T cells, not the APC. We incubate the APC with mitomycin C for 45 minutes to cross link their DNA, preventing proliferation. If you have a radiation source, irradiation can be used instead.
- Incubate antigen-presenting cells with peptide. Depending on the T cell line, a concentration of 0.1-5 ug/mL for 30 minutes is sufficient.
- Add T cells. We usually add 20,000 T cells per well of a 96 well plate with an equal number of APC.
- Incubate. Depending on what you want to measure, the incubation period can be 24-48 hours for cytokines or 3-4 days for proliferation.
- Read out activity. Culture medium can be collected after 24 hours. If you are testing CD4+ T cells, you can read out proliferation after 3-4 days.
Testing of Peripheral Blood Mononuclear Cells
We test our PBMC and evaluate their quality after thawing to simulate the conditions in which you will receive and use them. We check the cell count, viability, and cell surface antigens. The cell surface antigens allow us to characterize the cell types and proportions in the vial. We also measure the responses to several in vitro stimuli.
We use tetanus toxoid to measure T cell reactivity to antigens commonly vaccinated against. We also use the cytomegalovirus (CMV) antigen, although only chronically infected donors will respond. Lipopolysaccharide (LPS) measures the function of the monocytes in PBMC, and PHA activates all T cells regardless of their antigen specificity.
For more tips, see our Recall Antigen Testing protocol.
With all this information in hand, you can begin experimenting with confidence knowing the cells in each lot are fully functional.