I'm trying to make a Treg supression assay, to evaluate de ability of autologous Treg to inhibit the proliferation of Teff after treatment of patients with our product. To differentiate autologous monocytes into APCs would take me one week and I cant wait that long to do the assay.
So, Can I use human CD14+ freshly purified monocytes (without differentiating them) as APC in a Treg supression assay? Can someone help me with this trouble or give me a new idea? In addition, should they be irradiated?
Thanks
Hi Wendy
Don't irradiate, just use TGF-beta and IL-10 to decrease activation status and skew the T cells to a Treg profile. Remember, mono/mac ability to induce T cell antigen specificity is 2-3 logs lower than DC. Their ability to suppress Teff is going to be much less than DC2 with TGF-beta, so you might want to spend 4 days transforming your CD14+ to CD1/CD11+ DC2.
Cheers
Bruce
Hi, Wendy,
For Treg suppression assay, I usually used the nonTreg section as APCs when you isolate Treg through Miltenyi kit. Monocytes may not be a good APC. Alternatively, you can use B cells as APCs.
Hi
I had similar problems in T cells priming but i used to freeze the t cells till the CD14 are matured to be dendritic cells which usually takes a week.
so when the Dcs are ready i thaw t cells and prepare the co culture
The other choice as what Zhang said is to use EBV infected B cells (available in university of Liverpool)
Regards
Hi
Thanks to everyone for yours advices. Due to the few amount of blood that can collect from patient, it won't be a good idea freeze the T cell, 'cause I will loose to much cell during thawning process. So, I'm going to use the non Treg section as APC.
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