Mito fuel flex test kit

The cells' dependency on oxidizing three mitochondrial fuels (glucose, glutamine, long-chain fatty acids) was measured by determining the rate of oxidation of each fuel of cells in the presence or absence of fuel pathway inhibitors. According to the manufacturer's instructions, it was performed using a Mito Fuel Flex Test kit (Agilent).

A Seahorse Mito Fuel Flex Test kit and cell mitochondrial stress test kit were used to measure the oxygen consumption rates (OCR) in CD3⁺ T cells according to the manufacturer’s protocol (Agilent, Santa Clara, CA). Purified T cells were seeded in each well of an XFp cell culture mini plate at a density of 2 × 10⁵ cells/well. The XFp cell culture plates were pre-coated with CellTak (Corning, NY, USA) to allow the T cells to adhere to the bottom of wells. Measurements were performed on a Seahorse XFp Analyzer (Agilent, Santa Clara, CA, USA). The Seahorse Wave software was used to interpret the acquired data and calculate the OCR of the purified T cells.