recently , i have found an interesting phenomenon when i did some experiment about murine Treg. my protocol as follows:
first of all i use cell sorting kit isolate cd4+cd25+cells(i consider them as Treg) from murine spleen;
then i use 1uM cfse label these Treg cells and add 50000 cells /200ul into 96 well round bottem plates with soluble anti-cd3(1ug/ml)and IL-2(100IU) as stimulation(medium control); in another group, i use same cell concentration and use soluble anti-cd3(1ug/ml), IL-2(100IU) and gp96, a heat shock protein as stimulation;
then i culture these cells for 4d and detect the proliferation of Treg and IL-10 expression；
I found that Treg can proliferate in both stimulatin, but only latter group that added gp96 could significantly enhance IL-10 expression level.
my question is :
1.why only use soluble anti cd3(1ug/ml) and IL-2(100IU/ml) without add cd28 could induce Treg proliferation?
2. is there any protocol that i can sustain treg in vitro ,by that i mean a culture condition that Treg don't proliferation or die.and then i plan to add gp96 again and detect the expression level of IL-10 and further analyze the impact of gp96 on Treg function.
i hope everyone can help me solve my puzzle. thank u very much !!
In terms of your first question, you can find some clue from this article.
CD4+CD25+ Immunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production.
I believe stimulating CD28 might result in more non Treg CD4+ cells.
If you are interested in measured amounts of Tregs in each well, you can culture and induce big amount in a flask, and then sort for (CD4+CD25+CD120-) and distribute an equal amount of relatively concentrated culture of Tregs.