PMC:2800179 / 4645-6641 JSONTXT

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After neutralization with NaOH 4N, the solution was ultra filtered through a hollow fibre cartridge with MW cut-off 5 kDa, (Amicon Inc, Beverly, USA). For the medium molecular weight fraction, the carrageenan solution was hydrolyzed with 0.3% (v/v) concentrated sulphuric acid for 30 min at 60°C. After neutralization, the supernatant was ultra filtered (MW cut-off 100 kDa). The filtrate was submitted to a second ultra filtration (MW cut-off 5 kDa). Both preparations of dCGN were precipitated with 4 volumes of 95% ethanol, dried at room temperature and ground to small particles (1 mm in diameter). Using gel-permeation chromatography in combination with light scattering measurements (see Viebke et al. [21]), it was confirmed that the low fraction had an average molecular weight of 10 kDa, and the medium fraction of 40 kDa. The sulphate content of polysaccharides in both fractions was measured following the method of Quemener et al. [22]. Finally, the absence of polysaccharide structure modifications in the two fractions was confirmed using 2H-NMR spectroscopy. The absence of LPS contamination in the two fractions was confirmed using the e-Toxate® kit (Sigma, St Quentin Fallavier, France). Before use in cell culture, the two fractions were dissolved in complete medium during 30 min at 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