What concentration of anti-CD3 and anti-CD28 Ab do you use for coating? Which buffer do you use and at which pH? How many T cells do you plate for the proliferation assay?
The coating I do in 96-w flat bottom plate with 5 ug/mL aCD3 in PBS 1x (pH 7.4) overnight @ 4°C. The day after I plate 50'000 naïve T cells (CFSE stained) and I add 1 ug/mL of soluble aCD28. You can then measure proliferation by FACS at days 2,3,4,5 (CD8 cells proliferate faster than CD4). Hope that helps, good luck
whereas the anti-CD3 has to be coated (and the best you could do is to titrate the antibody concentration on a plate), the anti-CD28 can be added soluble (you will bet a higher proliferation if it is soluble). Amounts..... titrate both, you can get very nice sigmoideal curves and from there the appropriate amounts of antibody (most likely you are using a big excess of both and this is expensive).
I much appreciate your answers and suggestions. I should titrate all combinations of cell numbers, antibodies, and so on.
I wonder, how many CPM (if you work with thymidine) you obtain with 0.5x10^5 cells/well of flat-bottomed 96 plate.
It seems to me that I should use at least 2x10^5 cells, since 0.5x10^5 T cells occupy less than half of a well.
I use round-bottom instead of flat-bottom well plates with immobilized anti-CD3 (10 µg/ml, 50 µl per well, 4 °C overnight), soluble anti-CD28 1 µg/ml (0.5 µg/ml work as well) and (if required) 50 IU/ml IL-2.
I have obtained good results using the BD anti-CD28 anti-CD49d cocktail, which is supplied 1000X and can be used soluble.
That's interesting..Presumably, you do not have Tregs in your responders then
An important point which has not been mentioned till now is also which antiCD3 monoclonal antibody are you going to use.
At the same concentrations different ones can induce very different proliferative responses depending on which epitopes they target and how they target it.
The problem with Tony's protocol is that if you use round-bottom plates the amount of actual CD3-coated to the plastic that can get in contact with the T cells is relatively small since the cells stick together. If you use flat bottom you do not need to add exogenous IL2
Thank you for the discussion. Can anyone please tell me, what informations can we get by the proliferation assay if i want to use as a positive control? I am plating 1million cells/well with four different conditions (3 stimulants and 1 unstimulated). Want to use one more well as proliferation control with Anti CD3, anti CD28. There will be any increment in Treg cells or cytokine production? Please suggest. thanks.
4˚C you can do it if you coat ON the antibody, if you do it 1 or 2 hours should be at 37˚C
I coat 96-well flat bottom with1.5 ug/ml anti-CD3 overnight / 4˚C and then add 1 ug/ml anti-CD28 as I am adding my cell culture media and cells the following day.
why need to coat anti-cd3 antibody? should i directly add in the RPMI medium of PBMC?
Jiangnan He
Depends on the antibody you use. If you use the soluble antibodies activation will work too.