Cells were grown on galactose-CDM, glucose-CDM and in the nasal synthetic medium to an OD600 = 0.4 and treated with 250 μM spermine NONOate (or left untreated, the control sample), and harvested 1 and 3 h later. After centrifugation (11800 ×g for 2 min) and filtering (0.22 μm Pall filters), the supernatants were stored at -20°C and later used for the 1H-NMR studies. Extracellular metabolites were quantified on a Bruker AVANCE II500 MHz spectrometer (Bruker BioSpin GmbH) operated by TOPSPIN software using a 5 mm BBIXYZ high resolution probe head, at 16°C, and standard Bruker pulse programs. Spectra were referenced to the resonance of externally added trimethylsilyl propionate (TSP), designated at 0 ppm.
Resonances were assigned by addition to the supernatants of pure compounds and comparison with data available in the literature (Carvalho et al., 2011 (link)). Concentrations were calculated from the areas of the resonances of the 1H-NMR spectra considering the TSP resonance area, and using an appropriate resonance saturation correction factor (Carvalho et al., 2011 (link)). The metabolites concentration was normalized to the OD600, measured for each condition.
Resonances were assigned by addition to the supernatants of pure compounds and comparison with data available in the literature (Carvalho et al., 2011 (link)). Concentrations were calculated from the areas of the resonances of the 1H-NMR spectra considering the TSP resonance area, and using an appropriate resonance saturation correction factor (Carvalho et al., 2011 (link)). The metabolites concentration was normalized to the OD600, measured for each condition.
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