Monocyte-derived macrophages were differentiated with LPS/IFNγ and cultured for 24 hours. Alpha-Ketoglutarate Colorimetric/fluorometric Assay Kits (Bio Vision, K677), Glutamine Assay Kits (Colorimetric) (Abcam, ab197011) and Glutamate Assay Kits (Fluorometric) (Abcam, ab138883) were used to quantify extracellular glutamine, intracellular glutamate and intracellular αKG.
For tissue glucose measurement, synovial tissue samples were used immediately after harvest. The tissues were weighed, rapidly extracted with 1mL deionized water for 15 minutes at room temperature. Glucose in tissue lysates was quantified applying enzyme based high-sensitivity Glucose (HK) Assay kits (Sigma-Aldrich).
For tissue glutamine measurement, synovial tissues (10 mg) were washed, transferred to a pre-chilled tissue homogenizer, and homogenized on ice using 10X(v/w) hydrolysis buffer. Homogenates were centrifuged at 10,000 g for 10 min, and supernatants were collected. Glutamine in tissue lysates was quantified applying Glutamine Assay Kits (Colorimetric) (Abcam, ab197011).
For tissue glucose measurement, synovial tissue samples were used immediately after harvest. The tissues were weighed, rapidly extracted with 1mL deionized water for 15 minutes at room temperature. Glucose in tissue lysates was quantified applying enzyme based high-sensitivity Glucose (HK) Assay kits (Sigma-Aldrich).
For tissue glutamine measurement, synovial tissues (10 mg) were washed, transferred to a pre-chilled tissue homogenizer, and homogenized on ice using 10X(v/w) hydrolysis buffer. Homogenates were centrifuged at 10,000 g for 10 min, and supernatants were collected. Glutamine in tissue lysates was quantified applying Glutamine Assay Kits (Colorimetric) (Abcam, ab197011).