Figure 6. Analysis of the olfactory neuroepithelium (ON) cell’s proliferation, differentiation and apoptosis. (A) Cells were incubated for 5 days under proliferative conditions, after which they were analysed by immunofluorescences (IF) or by flow cytometry (FC).(B) Quantification of proliferative Ki67+cells relative to the total number of cells (DAPI+).(C) Quantification of the total number of cells (DAPI+) in the culture. (D, E) Representative immunofluorescence images of cells in culture derived from control subjects and cannabis users. Cells under proliferation are immunolabelled in green (Ki67+) and nuclei are stained in blue (DAPI). Selected areas marked with dotted lines are showed in the white squares with the green labelling alone. (F)Quantification of differentiated NeuN+ cells relative to the total number of cells (DAPI+). (G-J)Representative immunofluorescence images of cells in culture derived from control subjects and cannabis users. Differentiated NeuN+ and GFAP+ cells are immunolabelled in green and nuclei are stained with DAPI (blue). Selected areas marked with dotted lines are showed in the white squares with the green labelling alone. (K-L) Representative flow cytometry plots of cells in culture derived from control subjects and cannabis users. (M) ON cells were labelled with Annexin V (A) and Propidium Iodide (PI) followed by flow cytometric analysis to measure apoptosis. The percentage of the following cell populations is indicated: A-/PI- are viable cells (lower left quadrant); A+/PI- are early apoptotic cells (lower right quadrant); and A+/PI+ are late apoptotic cells (upper right quadrant). (N) Quantification of the percentage of cells in early (A+/PI-) and (O) late (A+/PI+) apoptosis. Scale bar = 50 μm. * p < 0.05; ** p < 0.001; (Student’s t-test , n=5).