Seahorse mitochondrial stress test assay

Metabolic rate of Tregs was determined via the Agilent Seahorse mitochondrial stress test assay modified with an initial palmitate injection (Santa Clara, CA). Briefly, expanded Tregs were cultured for 4 hours in substrate-limited medium (Kreb’s/HEPES Buffer) before being adhered to a Agilent Seahorse XF96 cell culture microplate with Cell Tak (Corning, Corning, NY) at a density of2.5×10⁵ Tregs/well which were then washed with fatty acid media (the day of the assay in a 96-well assay plate)(Wang et al., 2013 (link)). The stress test for Tregs was performed by injection of Palmitate-BSA before the injections of all standardized reagents for the mitochondrial stress test as described in the protocol. CCL22 (Peprotech) was injected at a final concentration of 100 ng/ml to measure ECAR over time. All reagents for the mitochondrial stress test, including Palmitate-BSA and BSA controls, were purchased from Agilent.