Agilent poroshell 120 c18

To test if the addition of SIM affects the intracellular levels of TAM, cells were seeded at a density of 2×10⁴ cells/well in 24-well plates and left for 24 hours. Cells were incubated with TAM alone and with SIM, medium was then aspirated at 0, 2, 4, 6, 24, and 48 hours, centrifuged, and the supernatants were used for assay. Two hundred microliters of the supernatant were mixed thoroughly with 200 µL acetonitrile (Alliance Bio, USA) and centrifuged at 1,400 rpm for 15 minutes at 4°C. The clear supernatant was injected into an AB SCIEX LC/MS/MS system (AB SCIEX 3200 QTRAP, Germany) adapting the method for TAM determination.27 (link) The system is equipped with an electrospray ionization (ESI) source and an Agilent 1260 affinity HPLC system, consisting of a vacuum degasser, a binary pump, and an autosampler to determine the concentration of TAM. Analyst 1.5.2 software was used for data acquisition and processing. The analytical column used was Agilent Poroshell 120-C18 (50 mm×3 mm×2.7 µm, Agilent, Germany) at 25°C. The mobile phase consists of 0.1% formic acid/water (solvent A) and 0.1% formic acid/acetonitrile (solvent B), delivered at a flow rate of 0.5 mL/min. Mass spectrometric analysis was performed in the positive ion mode.

To study whether the addition of SIM can affect the level of TAM, the level of TAM alone and in its combination with SIM were determined using LC/MS/MS. Cells were seeded at a density of 2 × 10⁴ cells/well in 24-well plate and left for 24 h. Cells were incubated with TAM alone and with SIM, the medium was then aspirated at 0, 2, 4, 6, 24 and 48 h, centrifuged and the supernatants were used for the assay. Two- hundred microliters of the supernatant were mixed thoroughly with 200 μL acetonitrile (Alliance Bio, USA) and centrifuged at 1400 rpm for 15 min at 4∘ C. The clear supernatant was injected into AB SCIEX LC/MS/MS system (AB SCIEX 3200 QTRAP, Germany) adapting the method of Gjerde et al. [25 (link)] for TAM determination. The system is equipped with an electrospray ionization (ESI) source and an Agilent 1260 affinity HPLC system, consisting of a vacuum degasser, a binary pump, and an autosampler to determine the concentration of TAM. Analyst 1.5.2 software was used for data acquisition and processing. The analytical column used was Agilent Poroshell 120-C18 (50 mm × 3 mm ×2.7 μm, Agilent, Germany) at 25^∘ C. The mobile phase consists of 0.1% formic acid/water (solvent A) and 0.1% formic acid/acetonitrile (solvent B), delivered at a flow rate of 0.5 mL/min and the analysis was performed using the positive ion mode.