Hello,
I will start performing MLR using lymphocytes from mice's spleen and I would like to know the better way for the stimulation. If is using PHA-P or CD3/CD28?
I would like to know as well if is better use PBMC from blood or spleen?
Best regards,
Mostly optimum concentration recommended PHA for stimulation of whole blood is 5ug/ml for 5 days.
The following article although been published two decades ago might be helpful for your reference.
Dear Aline D. de Lima .
To your first answer, PHA-P also gives good results for stimulation as well as proliferation at different doses (5ug/mL - 10ug/mL) stimulation from 24-72 hrs while proliferation upto 72 hrs.
Anti-CD3/CD28 beads are best if you may you. As excess dose of PHA is cytotoxic (does of the PHA is to be optimized in your lab, which will take much time) while does of Beads are predefined (may be you need to do fine tune) usually.
To your second, If you need to perform experiments on T cells than Spleen will be good sources of T cells while blood constitute heterogenic cell population. You may proceed with either source but if you can get spleen (in case at the end point of experimet you does not need that animal) otherwise blood will also give good results.
Dear Aline,
I'm not sure I understan your 1st question: are you asking what is the best choice to stimulate mouse T cells among MLR, PHA or CD3/28?
The answer to that is CD3/28, as it is a generic pan-T cell stimulation that activates most cells in the culture. However, the choice of activation modality depends on the scientific question you wish to answer. I can advise more acurately, if you mention your question (btw, for mouse, use ConA rather than PHA).
For the 2nd question the term PBMC (Peripheral Blood Lymphocytes) only refers to blood circulating leukocytes, while those in spleen are refered to as "splenocytes" as they are different. Most people use spenic lymphocytes, as they are more mature and easier to obtain and faster to isolate.
Hope it helps.
You are asking two questions. The best mitogen for murine T-cells is Con-A. Optimal is about 5 ug/ml.
A MLR differs as it is a T-cell response to allo antigens typically using irradiated spleen cells from another mouse strain. Example C57bl/6 vs Balb/c. A 3 to 10 ratio of responders to 1 simulator is frequently optimal. Best to do a dose response first.
If you want to measure a T-cell response, a mitogen is easiest, followed by anti CD3 and CD28 and lastly a MLR. Note, all are easy. A mitogen is also the cheapest.