Lc 321 322 350 pump

MDA-MB-231 cells were seeded at a density of 20 × 10⁴ cells/well in a 6-well plate and transfected with the control or GLI1 siRNA at a concentration of 20 nM. After 48 h, the cell medium was collected and mixed by vortexing with acetonitrile containing the internal standard (IS) thiamine. After centrifugation at 4000 rpm for 5 min, the supernatants were collected and analyzed. Lactate content in the cell media was measured using HPLC. The liquid chromatography (LC) system used in this study comprised a Gilson pump (LC-321 322 350 pump), autosampler (Gilson-234), and UV (UV/VIS-151) detector (Gilson, France). Detection and quantification were performed on a C18 column (Synergi 4 µm Hydro-RP 80 Å, LC Column 250 × 4.6 mm) preceded by a pre-column (Phenomenex, USA). The isocratic mobile phase of water with 0.1% phosphoric acid was used for the analysis (0.8 mL/min flow rate, RT). Lactate concentration was measured using a UV detector at 210 nm.

The 3-IS content was estimated in the serum, urine, and kidney tissues using high-performance liquid chromatography (HPLC). The extraction of the samples was performed using 70% acetonitrile, an organic solvent, in a multi-step process. The extracted samples were mixed thoroughly with 2-naphthalene sulfonic acid used as an internal standard, and measured using HPLC (Gilson, LC-321322350) at 280 nm. The retention time of 3-IS was 6.8 min, and the extraction recovery was 84%. The HPLC apparatus used in the present study comprised a Gilson pump (LC-321322350 pump), an autosampler (Gilson-234), and a UV (UV/VIS-151) detector (Gilson, France). A C18 column from Agilent (Torrance, CA, USA) with a pre-column (250 mm 4.6 mm, and 5 m) was used for detection and quantification with a flow rate of 0.9 mL/min at room temperature. A 70:30 v/v combination of acetonitrile and 0.1% trifluoroacetic acid in Milli Q water was employed as the isocratic mobile phase.

Liquid chromatography (LC) system included a UV/Vis-151 detector (Gilson), an autosampler (Gilson-234), and a LC-321/322/350 pump (Gilson, France). Detection and quantification were performed using a Synergi Hydro-RP C18 column (250 × 4.6 mm, 4 μm, 80 Å; Phenomenex, USA). The flow rate was maintained at 0.8 ml/min for lactate. Isocratic mobile phases included 0.1% phosphoric acid in water for lactate. After extraction, the samples were transferred to a sample tube with acetonitrile containing thiamine (internal standard) and mixed thoroughly. The samples were centrifuged for 5 min at 1503×g. The supernatants were analyzed by performing HPLC (LC-321/322/350 pump) at 210 nm for lactate.