Vascular smooth muscle cell line (A7r5) was obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin (Life Technologies). Cells were maintained at 37°C in an atmosphere of 5% CO2 with the medium replaced every 24 hours.
1.5×107 cells were treated with either S- or R-Carvedilol at a concentration of 5 µM due to MTT reduction analysis [10] (link). Cells treated in parallel with equal amounts of dimethyl sulfoxide (DMSO) were used as control. After 48 hours, culture medium was collected, filtered through a 0.2 µm filter (WHATMAN), and immediately frozen in liquid nitrogen. Cells were washed with cold PBS twice, harvested and snapped frozen in liquid nitrogen. Carvedilol enantiomers were previously separated from their racemic form using High Performance Liquid Chromatography (HPLC) in our lab [10] (link). Four independent experiments were conducted.
The extraction of intracellular metabolites from A7r5 cells was performed using a modified Mainak Mal [41] (link) and Hindrik Mulder [11] (link) procedure. Cell pellets were dissolved in a mixture of chloroform/methanol/water in ratio of 2∶5∶2 (v/v/v). Ribitol (2 mg/mL dissolved in water, 10 µL) was added into the extraction solvent as internal standard to correct for metabolites losses during sample preparation. The samples were ultra-sonicated in an ice bath ultra-sonicator for 30 min and subsequently centrifuged at 18000 g for 10 mins at 4°C. 0.8 mL supernatant was collected and evaporated to complete dryness overnight using a TurboVap® LV Concentration Workstation. The culture medium samples were prepared by spiking 500 µL of each culture medium with 10 µL internal standard solution (ribitol, 2 mg/mL dissolved in water) and lyophilised. Immediately prior to GC-MS analysis, derivatization was performed in two steps: Firstly, methoximation was carried out by dissolving the sample in 50 µL of 20 mg/mL solution of methoxyamine hydrochloride in pyridine to protect the carbonyls. The incubation was kept at 37°C for 60 min. Silylation was then carried out by adding 100 µL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) to each sample for 30 min at 70°C. After incubation, samples were shaken for 2 hours at room temperature and then transferred to vials for GC-MS analysis.
1.5×107 cells were treated with either S- or R-Carvedilol at a concentration of 5 µM due to MTT reduction analysis [10] (link). Cells treated in parallel with equal amounts of dimethyl sulfoxide (DMSO) were used as control. After 48 hours, culture medium was collected, filtered through a 0.2 µm filter (WHATMAN), and immediately frozen in liquid nitrogen. Cells were washed with cold PBS twice, harvested and snapped frozen in liquid nitrogen. Carvedilol enantiomers were previously separated from their racemic form using High Performance Liquid Chromatography (HPLC) in our lab [10] (link). Four independent experiments were conducted.
The extraction of intracellular metabolites from A7r5 cells was performed using a modified Mainak Mal [41] (link) and Hindrik Mulder [11] (link) procedure. Cell pellets were dissolved in a mixture of chloroform/methanol/water in ratio of 2∶5∶2 (v/v/v). Ribitol (2 mg/mL dissolved in water, 10 µL) was added into the extraction solvent as internal standard to correct for metabolites losses during sample preparation. The samples were ultra-sonicated in an ice bath ultra-sonicator for 30 min and subsequently centrifuged at 18000 g for 10 mins at 4°C. 0.8 mL supernatant was collected and evaporated to complete dryness overnight using a TurboVap® LV Concentration Workstation. The culture medium samples were prepared by spiking 500 µL of each culture medium with 10 µL internal standard solution (ribitol, 2 mg/mL dissolved in water) and lyophilised. Immediately prior to GC-MS analysis, derivatization was performed in two steps: Firstly, methoximation was carried out by dissolving the sample in 50 µL of 20 mg/mL solution of methoxyamine hydrochloride in pyridine to protect the carbonyls. The incubation was kept at 37°C for 60 min. Silylation was then carried out by adding 100 µL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) to each sample for 30 min at 70°C. After incubation, samples were shaken for 2 hours at room temperature and then transferred to vials for GC-MS analysis.
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