Fig. 5
From: Crosstalk between tumor cells and lymphocytes modulates heparanase expression
Exosome analysis. The exosomes were purified from collected medium of a healthy woman lymphocytes, co-culture lymphocytes (lymphocytes and MCF-7 cells) or MCF-7 cells, respectively, non-activated medium, MCF-7 medium and activated medium. The exosomes were immobilized in glass slides by cytocentrifugation and subsequently permeabilized with 0.01% saponin and analyzed by confocal microscopy after anti-heparan sulfate and anti-CD63 labeling. Heparan sulfate was revealed using a secondary antibody conjugated with Alexa Fluor®647 (red fluorescence), while exosomes were labelled using anti-CD63 conjugated with FITC (green fluorescence). The ROI images correspond to 1.45 × zoom. The values expressed in the graphs indicate the number of exosomes labelled with heparan sulfate antibody (red) and anti-CD63 (green). Bars in wide field images: 5 μm. ROI: region of interest. Statistical analysis was performed using One-Way analysis of variance with Dunnett´s multiple comparison test. */*** number of exosomes (Heparan sulfate labelling) comparing activated lymphocytes to the non-activated lymphocytes and MCF-7 medium to the non-activated lymphocytes, */***p < 0.05. */** number of exosomes (CD63 labelling) comparing activated lymphocytes to the non-activated lymphocytes and MCF-7 medium to the non-activated lymphocytes, */**p < 0.05. The experiment was performed using lymphocytes obtained from one healthy donor which is shared in two samples, lymphocytes non-activated and lymphocytes activated with MCF-7 cells (co-culture lymphocytes)