Using Seahorse Technology as an Efficient Way of Verifying T Cell Stimulation.

Antibody-dependent stimulation is commonly used to activate, differentiate, and/or to expand T cells for downstream analysis and functions. Such stimulations are inherently connected to endogenous phosphorylation which is commonly assessed using various methods including immunoblotting. However, antibody-dependent stimulation of T cells is also inherently connected to changes in endogenous metabolic activity. We describe methods used to stimulate T cells using soluble and immobilized antibodies in conjunction with immunosuppressive cyclic AMP (cAMP). The stimulations were assessed by downstream phosphorylation using immunoblotting and metabolic changes using Seahorse technology. We use phosphorylation of ERK 1/2, extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) as readouts to represent the activation state of T cells. We also describe how these methods can be used to assess inhibitory stimuli exemplified by the cAMP analogue 8-(4-Chlorophenylthio) adenosine 3',5'-cyclic monophosphate (8-CPT-cAMP), but only if 8-CPT-cAMP is added before anti-CD3. Together the methods described in this chapter provide a comprehensive guideline to isolate, stimulate, and assess T cell stimulation. Moreover, we demonstrate the Seahorse technology as a time and work-efficient way of assessing the effects of T cell stimulation in real time.