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.