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).