Citrate assay kit

α-Ketoglutarate assay kit (Cat: K677-100, Biovision, Milpitas, CA, USA), citrate assay kit (Cat: K655-100, Biovision, Milpitas, CA, USA), and glutamine assay kit (Cat: K556-100, Biovision, Milpitas, CA, USA) were used to measure the intracellular α-KG, citrate, and glutamine level, respectively. Isolated HUVECs or endothelial cells were resuspended in an assay buffer. Following sonication and centrifugation, the supernatants were used to analyze the α-KG, citrate, and glutamine level according to the manufacturer’s instructions.

HepG2 cells were seeded at 2 ×10⁵ cells per well in 24-well culture plates and cultured in high-glucose medium for 3–4 days. After 24-h treatment with AICAR or metformin using a 5 mM glucose concentration, cells were exposed to physiological citrate concentration (200 µM) citrate in a NaCl uptake buffer (140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, and 5 mM glucose, buffered with 25 mM Hepes/Tris, pH 7.5) for 30 min. Cells were then washed with NaCl buffer and 200 µL of 100% ethanol was added to each well to lyse the cells and precipitate proteins. The plate was then kept in an incubator set at 37 °C to evaporate alcohol. The contents in the wells were then collected using 100 µL of nuclease-free water and spun down at 14,100 rpm for 30 min. The supernatant was collected and used for estimation of citrate using the BioVision citrate assay kit (San Francisco, CA, USA).

Concentration of citrate and alpha-ketoglutarate (α-KG) in whole-cell lysates from preactivated CD4⁺ T_EMs was measured by fluorescence assay using a kit according to the manufacturer's instructions (The Citrate Assay Kit: ab83396, α-KG Assay Kit: ab83431; Abcam). Relative fluorescence units/intensity were quantified using a Varioskan Flash Multimode Plate Reader (Thermo Scientific).

Intracellular citrate levels were detected by the Citrate Assay Kit (Abcam, Biomed Diagnostics (TH) Co., Ltd, Bangkok, Thailand). Briefly, HepG2 cells were seeded in a 60‐mm³ petri dish and allowed an overnight period of attachment. Citrate was formed by the addition of oxaloacetate to the acetyl group of acetyl‐CoA derived from the glycolysis pathway. Citrate was converted to pyruvate which was then quantified by fluorescence intensity measured at Ex/Em 535/590 nm using the Synergy HT Microplate Reader with gen5 data analysis software (Biotek Instruments, Inc.). Results were expressed as a percentage of intracellular citrate compared with the control.

The important substrate for the de novo synthesis of fatty acids is cytosolic citrate which can be quantified by the conversion of citrate to pyruvate via oxaloacetate. The pyruvate product was measured by colorimetric or fluorescence methods. The citrate assay was performed using the Citrate Assay Kit (Abcam) as described in the manufacturer’s protocol. Briefly, HepG2 cells were plated at a density of 1×10⁶ cells and allowed to attach overnight. Cells were incubated with capsaicin at concentrations of 0.5 and 1 mM for 24 h. After treatment, cells were harvested and homogenized with citrate assay buffer and then pelleted by centrifugation. The citrate standard and cell sample were converted to pyruvate, followed by adding the reaction mix containing a fluorescent probe and developer, and further measured fluorescence at Ex/Em535/590 nm using the Synergy HT Microplate Reader (BioTek Instruments) with Gen5 Data Analysis software. Results were expressed as the percentage of intracellular citrate compared with the control.

Culture supernatant from BMDMs was collected as indicated in the experiments and the cytokine concentrations were measured by commercial ELISA kits according to the manufacturer’s instructions (BioVision). Lactate levels in media were quantified by using a lactate lit (K607 Lactate Colorimetric/Fluorometric Assay Kit, BioVision). The intracellular concentrations of acetyl-coA, citrate and pyruvate in BMDMs were detected by using the PicoProbe Acetyl-coenzyme A assay kit (ab87546, Abcam), the citrate assay kit (ab83396, Abcam) and the Pyruvate assay kit (K609, BioVision), respectively. For NAD and NADH measurement, total NAD and NADH levels were measured using a NAD/NADH Quantitation Colorimetric Kit (K337, BioVision). The ratios of NAD⁺ (NAD–NADH) to NADH were calculated. All experiments were performed at least three times and the data were normalized by the cell numbers or protein contents.