So you re-stimulate in vitro ex vivo +/- antigen or CD3 or polyclonal

for 2, 4, 6 h

than you fix and stain and analyze the cells

Dead cells ex vivo make no cytokines in vitro

dead is dead

Thx Thilo,

I think your answer help a lot.

Also, what do you think are the assumptions needed to validate that the relative cytokine level will still be what it was in vivo upon ex vivo re-stimulation? Like, 1. ex vivo cells will produce cytokines in the same way as stimulated in vivo, 2. the cytokine transportation inhibition will effect without altering relative cytokine level remaining inside cells, 3. centrifugation causes cell lost evenly in cytokine-positive and -negative group...

jiyuan

as soon as you get a cell out of the body (blood, mouse, etc), you will perturb their physiology. But everything being rendered equal in your experiments, what you really need are good controls. What an ex vivo restimulation test really tells you are differences between controls and tests, not the absolute reality nor quantity of cytokines in vivo. I will add PMA/iono for 4 hrs as a restimulation agent although it will perturb phenotype (CD3, CD4)