I need to stimulate CD8 T cells to check their proliferation by CFSE staining. Is it a good idea to add the soluble CD3 antibody to the co-culture of CD8 T cells and irradiated APCs to check the proliferation by CFSE staining or it is better to use plate-bound anti-CD3 antibody for CD8 T cell stimulation?
Use the bound anti CD3, combined with bound anti cd28.
soluble Ab will block the signaling leading to the opposite effect
bound anti-CD3 is enough, you do not need the anti-CD28. That said we use anti-CD3 dynabeads from invetrogen in our CFSE assay.
http://www.lifetechnologies.com/order/catalog/product/11151D?ICID=search-11151d
CD3 needs to be cross-linked, so you may either plate aCD3 Ab overnight at 4C (or just 2hours at 37) in PBS, remove and wash, then add cells. Otherwise, use DynaBeads PanMouse kit as suggested by Sarah (be sure that your aCD3 has a mouse Fc).
CD28 can be used in a soluble form.
Good luck,
I have routinely used soluble anti-CD3 and anti-CD28 to stimulate CD8 cultures derived from the spleen and lymph nodes of mice. In my experience, soluble anti-CD3 actually gives better activation when compared to plate bound at the same concentration. I do use plate bound anti-CD3/28 when I intend to inject the cells into mice shortly after activation for fear of activating complement.
Thank you Hu Zeng. I have to co-culture purified CD8 T cells with APCs and measure proliferation by CFSE method (FACS), so I should use soluble anti-CD3 then. But do I need to add anti-CD3 along with anti-CD28 or add anti-CD28 after some incubation time?
Hi Tabasum,
I have a few questions. If you are looking at antigen specific proliferation, then the APCs should be sufficient to induce proliferation of your T cells ( APCs can provide signal 1 (TCR), Signal 2 (co-stimulatory CD28) and Signal 3 (cytokines) requried for T cell proliferation. I am not sure what the experimental hypothesis is when you use both APCs and anti-CD3. The reason you use anti-CD3/CD28 is when you are looking for polyclonal (not antigen specific) expansion of T cells. Once again, if you have any further questions, do not hesitate to inbox me. Once i have a better idea of what the experimental question is, then i can be able to help with more details.
simon.
Hi Simon,
I think you are right. I am messing up my experiment by adding APCs to anti-CD3 coated plate. All I need to check at this stage is to see whether my CFSE dilution is working or not. So, I think it is better if I just coat my plate with anti-CD3 to check polyclonal expansion of T cells. Once I feel that everything is working, I can use APCs without coating plate with anti-CD3 in my real experiment in which I have to load cells with peptide. I really appreciate your suggestion.
Thanks,
Tabasum
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