We are currently using the FASCIA method for in vitro stimulation of whole blood (ref: Marits P. et al. Clin Immunol PubMed ID 24909732). We use PHA, PWM and ConA for stimulation but I don't really understand where the differences in responses come from with these mitogens. Some times the same donor has a perfect response to PHA but only a moderate response to ConA. Does anyone happen to know why?
We'd like to have a stimulant that would be TCR specific but the CD3-CD28-antibody stimulation does not work in this setting since the incubation time is very long and the cells downregulate CD3 making it impossible to gate on them in flow cytometry analysis. Any recommendations for CD3-CD28 replacement that would still be TCR-specific and give robust responses?
All input is greatly appreciated!
Hi
Look this link PLZ
T cell stimulation and expansion using anti-CD3/CD28 beads. - NCBI
www.ncbi.nlm.nih.gov/pubmed/12667688
by A Trickett - 2003 - Cited by 116 - Related articles
T cell stimulation and expansion using anti-CD3/CD28 beads. Trickett A(1), Kwan YL. ... Following appropriate stimulation, T lymphocytes will proliferate extensively in vitro. Traditionally, mitogenic lectins such as phytohemagglutinin (PHA) and concanavalin A (Con A) have been used for polyclonal T cell stimulation. A more ...
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Hi Eliisa,
We used To work With ConA to stimulates murine lymphocytes but it was not working appropriately. We change it to PHA and it seems that, at least for us, PHA works better tha ConA.
regarding the CD3 staining, you should remember that once T cells is activated the cd3 is internalized, son the staining should
Thank you all for the answers! I'll probably try SEB, even though our long incubation might prove problematic.
Hi Eliisa,
Superantigens are Vb restricted so you might want to use a cokctail (SEB, SEA, SEE, TSST-1) to cover a large Vb spectrum. And of note, you need Ag presenting cells when you do your stimulation or else the superantigens won't work.
Good luck,
G.
We use whole blood so there should be both monocytes and B-cells acting as APCs.
Hei Eliisa!
Besides being a specific antigen, Candida albicans has proteins containing some superantigen-like properties. It has been a very reliable control antigen in my experiments. It induces peak-level proliferation after 5d incubation.
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