Hi everybody,
I've been growing T cells from a flow cytometry CD3-sorting for a long time, obtained from C57BL6 mice spleens. At least three hours before seeding, the p96-well plate is coated with aCD3e and aCD28 for allowing policlonal stimulation of the T-cells, but I have recently found that the T-cells are not being stimulated, without an apparent reason (I have not changed anything). Do you know any reason for this absence of stimulation? The animals, the culture medium and the procedure is exactly the same.
Thank you in advance!
Hi Carolina.
There doesn't seem to be an apparent reason for why this is happening. I woud guess that something may be goig wrong when attempting to coat your plate with the antibodies, do they not "stick" for some reason?
The other issue may be that your sorted cells may have damaged receptors. It is a very rare condition but it may have happened or have they lost expression of the relevant receptors? May be one way would be to re-facs your cells before adding them to culture.
I would also suggest using coating anti-CD3 and soluble anti- CD28 as these are considered mor potent in stimulating T cells in culture and also try to use different combinations of your antibodies until you hit the right one.
I would also attempt to grow your cells using anti CD3 and IL-2. If they grow well then there may be something with your CD28 ab not working or the cells have lost the CD28 signalling.
These are some suggestions, but may be proven helpful.
Hope i helped.
BW
Nikos
1) You had better check the viability of sorted T cells by stimulating those T cells with Concanavalin A, another polyclonal activator of T cells. If they grow well, your purified T cells is fine. 2) You had better coat plates with antibodies for overnight. 3) If your sorted T cells are really pure, there is no antigen presenting cells. Under these conditions, T cells are not stimulated with Concanavalin A or combination of anti-CD3 and CD28. You should add back antigen presenting cells.
We have had this problem in the past. Here are several starting points:
- In our experience if you fail to wash away unbound anti-CD3 it actually ends up inhibiting proliferation. So coat the plate with anti-CD3 the night before . On the day of adding the CD3 T cells, perform atleast 5 gentle washes of the well with PBS to remove any unbound anti-CD3. Then you can add soluble CD28 along with your sorted T cells.
Try this and let me know how it goes. The other alternative is to use CD3/CD28 coated beads which you simply add to the culture at the same time as your cells.
Hi Carolina,
In my experience you can stimulate T cells with anti-CD3 antibody, proven that you let them enough time to recover CD3 expression. If you are sorting T cells with a labelled anti-CD3 antibody, perhaps it is blocking the interaction with the plate-bound anti-CD3; thus you should let T cells recover CD3 expression before stimulating.
My other guess would be the antibodies, have you verified that the antibody batches have not already expired? How do you check that your system works? As Toshimitsu said Concanavalin is an excellent control for assessing that your cells are viable and functional.
Thank you for all your answers. I have added PHA at different concentrations (I couldn't find concanavalin here around) and when analyzing the T cells for CD3, CD25 and CD69 we found that even the control conditions presented elevated CD69, so this could indicate a pre-activation state. The point is that I've been using this protocol for at least a year and the T-cells were always activated (assesed even at a simple glance of the plate), and for some reason now it is not working.
I will try to coat overnight with CD3, washed it several times and add soluble aCD28, and I have also ordered a new batch of CD28.
I'll keep you updated!!
Dear Carolina,
Another thing that might help, we use nontreated plates (not nunc-treated), and the antibodies stick better to the plate so the activation is stronger, it made a big difference, (3 hours are enough then)
Maybe you can also use CTV labelling so it becomes easier to compare different activation conditions, just by looking at proliferation.
Regarding Simon's comment, I was not aware of it. I naively wash only once and it is working, but in limiting activation conditions it is probably true,
Best
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