Fig 7. Confocal immunofluorescence of functional and cell maturation markers in the SN4741 cells at long term in culture.

Nuclear marker DAPI (blue-label), synaptophysin (SYP: red-label) and G-protein-regulated inward-rectifier potassium channel 2 (GIRK2: green-label) proteins immunofluorescence were localized in SN4741 cells cultured on plastic wells (A1-A4), baseline thermal-treated non-carbon (BTTN) film (B1-B4), NGLC 5-nm-thick film (C1-C4), NGLC 20-nm-thick film (D1-D4) and NGLC 80-nm-thick film (E1-E4). Colocalization (merge) as yellow-label and the specific protein markers related with functional differentiation is also quantifying (F). Brightfields images of BTTN (B5) and 5 nm (C5), 20nm (D5), 80nm (E5) NGLC films are located in the right column of the panel. Colocalization percentage of specific protein markers for functional differentiation SYP and GIRK2 was also calculated (F) by means of Pearson's correlation coefficient, as previously described by Dunn et al. (2011) using the Fiji-ImageJ software and the plugin ‘Colocalization Threshold’. Data are expressed as mean±standard deviation (SD, n = 6); **p<0.01 and ***p<0.001 vs control (cells cultured in plastic wells); ‘f’ = p<0,001 mark differences between NGLC samples and baseline thermal-treated non-carbon (BTTN) film.