2.9 Mass spectrometry analysis.
High-resolution mass spectra were acquired on a Bruker (Bruker Daltonik GmbH, Bremen, Germany) solariX XR Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR-MS) equipped with a 7T superconducting magnet and a MALDI source. Spectra were acquired with a Time Domain size of 16 mega-word, an accumulation time of 0.1 s and a mass range of 100-2000 m/z. Moreover, the average number of scans was set to 50. For the analysis, a laser power of 32% and a number of laser shots of 28 were assumed. 2,5–dihydroxybenzoic acid was used as a matrix, while a solution of this compound and pure snail slime (or AuNPs slime sample), in a 1:1 ratio, was prepared and analyzed. The mass measurement accuracy reached values of less than 0.1 ppm.
2.10 Cell viability determination .
The viability of NCTC2544 human keratinocyte and RAW 264.7 macrophages was determined by trypan blue staining. NCTC2544 cells (2.0×105) were seeded in 6-well plates and allowed to attach overnight. On the next day AuNP colloidal solutions were added at the indicated concentrations. 48 h later 20 μl of cells were aseptically transferred to a 1.5 mL clear Eppendorf tube and incubated for 3 min at room temperature with an equal volume of 0.4 % (w/v) trypan blue solution prepared in 0.81 % NaCl and 0.06 % (w/v) dibasic potassium phosphate. Viable and nonviable cells (trypan blue positive) were counted separately using a dual-chamber hemocytometer and a light microscope. RAW 264.7 cells (3.0× 105) were seeded in 6-well plates and allowed to attach overnight. On the next day cells were pre-treated overnight with 1μg LPS/mL. The day after the LPS was removed and the cells replaced with fresh media in presence of AuNPs-SS. 24h later viable and non-viable cells were counted as described fo NCTC2544. The means of three independent cell counts were pooled for analysis.