High-Performance Liquid Chromatograph (HPLC) was performed to measure kynurenine in the supernatants of the TGF-β 1-stimulated MDCK cells and of the unstimulated MDCK cells (control).
Supernatants were deproteinized by centrifugation at 5000 g (15 min at 4 °C) with 10% trichloroacetic acid (1:1, v/v), filtered in millipore 0.25 μm and 20 μL was injected into HPLC instrument equipped with UV detector (YL-9300; YL Instrument, Anyang, Korea). Data were obtained using a reversed phase column (LUNA RP-18, 25 cm × 4.5 mm; Phenomenex, Torrance, Ca, US), at room temperature. Separation was done in the following mobile phase: buffer sodium acetate 10 mM in MilliQ water (A) and acetonitrile (B): 0–1 min (20% B); 1.01–1.5 min (5% B); 1.51–8 min (4% B). The flow rate was kept constant at 1 mL/min and peaks were detected at 254 nm. All chemicals used in the analysis, such as acetonitrile and acetate buffer, were of HPLC grades and were purchased from Sigma and Merck.
A kynurenine standard curve was constructed (2.0 μM, 4.0 μM, 8.0 μM, and 16.0 μM). Injections were done in triplicate and kynurenine was detected by 254 nm UV. Linearity was observed in the concentration range 0.5 to 100 μM of kynurenine and the samples were quantified against the calibration standard curves, where y is the peak in Voltage (mV) and x the concentration in μM (y = 1.1×-0.0468 R2 = 0.998) and retention times of 2.1.
Supernatants were deproteinized by centrifugation at 5000 g (15 min at 4 °C) with 10% trichloroacetic acid (1:1, v/v), filtered in millipore 0.25 μm and 20 μL was injected into HPLC instrument equipped with UV detector (YL-9300; YL Instrument, Anyang, Korea). Data were obtained using a reversed phase column (LUNA RP-18, 25 cm × 4.5 mm; Phenomenex, Torrance, Ca, US), at room temperature. Separation was done in the following mobile phase: buffer sodium acetate 10 mM in MilliQ water (A) and acetonitrile (B): 0–1 min (20% B); 1.01–1.5 min (5% B); 1.51–8 min (4% B). The flow rate was kept constant at 1 mL/min and peaks were detected at 254 nm. All chemicals used in the analysis, such as acetonitrile and acetate buffer, were of HPLC grades and were purchased from Sigma and Merck.
A kynurenine standard curve was constructed (2.0 μM, 4.0 μM, 8.0 μM, and 16.0 μM). Injections were done in triplicate and kynurenine was detected by 254 nm UV. Linearity was observed in the concentration range 0.5 to 100 μM of kynurenine and the samples were quantified against the calibration standard curves, where y is the peak in Voltage (mV) and x the concentration in μM (y = 1.1×-0.0468 R2 = 0.998) and retention times of 2.1.