Fig 1.
Schematic illustration of the experimental protocol for monitoring of extracellular metabolites.
Conditioned medium collected from growing cells is analyzed by Raman spectroscopy using Ag nanostructured substrates. In the insets: an optical image of NIH/3T3 cells (left) and a SEM image of a typical electroless grown Ag nanoisland pattern (right).
Fig 2.
SERS efficiency of the fabricated Ag nanostructured surfaces.
(a) SERS spectra of R6G at 1 μM concentration on the Ag nanostructured substrate (red line) and on the Ag flat substrate (black line). (b) 2D SERS mapping centered at the R6G band at 615 cm-1. The measurements were done with 532 nm laser line, 100 μW power and 10 second acquisition time.
Fig 3.
Raman spectra of the 3T3 conditioned medium collected from one to four DIVs.
Raman spectra of DMEM culture medium collected at different DIVs of mouse embryonic fibroblasts NIH/3T3 cell line. DIV0 corresponds to complete DMEM without conditioning with cell culture. The measurements were done with 532 nm laser line, 1 mW power and 10 second acquisition time in liquid conditions. The red and green lines indicate the peaks with increasing and decreasing intensities, respectively.
Table 1.
Position and possible corresponding metabolite of the Raman peaks detected in the conditioned culture medium.
Raman peaks detected in the cell culture medium and modulated in the intensity during conditioning time are listed. For each of them, the position in the spectrum and the possible corresponding metabolite, in accordance with the literature, are reported.
Fig 4.
PCA of the Raman spectra from DIV0 to DIV4.
PCA of the collected Raman spectra grouped per day (a) and grouped per similarities (b) according to the algorithm developed in [44]. For each DIV, the Raman spectra acquired in all the four replicates are considered.
Fig 5.
Analysis of the intensity for multiple Raman peaks of the cell conditioned medium.
The normalized integral curve areas of peaks corresponding to different analytes present in cell culture medium are reported from DIV0 to DIV4. Each color corresponds to a different analyte.
Fig 6.
Analysis of the Raw 264.7 conditioned medium after LPS stimulation.
Raw 264.7 cells are stimulated with LPS for 24 hours. At 0, 6, 9, 15, 20, 24 hours, A) IL12p40 production was quantified by ELISA and B) Raman spectra of conditioned medium were collected. By PCA analysis, the spectra collected at each time point clusterize. C) The cluster centers for each time point are reported (left panel) as well as the trajectory described (right panel).