Fig. 4. Flow cytometric analysis of synaptosomes-like particles in P2 fractions from AD and DS subjects.

a Representative plots indicate the gating parameters, based on size (1–3 μm), used to quantify synaptosomes in P2 fractions from the parietal cortex of CTRL, AD, and DS subjects. b Numbers of particles within the size of synaptosomes were reduced from 2.5e⁺⁶ ± 2.7 e⁺⁵ in control (mean ± SEM; n = 5 biologically independent samples, 1 from each control subject, each value per subject is the average of two independent experiments) to 1.5 e⁺⁶ ± 2.2 e⁺⁵ in AD (n = 5 samples, 1 from each AD case) and 1.6 e⁺⁶ ± 1.6 e⁺⁵ in DS (n = 6 samples, 1 from each DS case). One-way ANOVA showed effect of diagnosis on particle count was significant, F (2,13) = 12.04, P = 0.0011. Post hoc analysis using two-sided Dunnett’s method (AD and DS vs CTRL) indicated that the average number of particles was lower in AD and DS as compared to control (P values in the figure). c, d Representative forward scatter plots for synaptosome-sized particles from an AD and a DS subject compared to the same control. Notice a shift to smaller size particles in AD and DS subjects compared to the control. e Plots showing the proportion of particles within the size range of synaptosomes in P2 fractions that were further identified as being small (1 μm < diameter (∅) < 2 μm), medium (2 μm = ∅) or large (2 μm < ∅ < 3 μm) sized for each group (group means ± SEM; based on c, d size distributions. See Supplementary Fig. S2 for gating strategy). One-way ANOVA determined that effect of diagnosis was significant for large (F (2,13) = 29.00, P = 1.61 e⁻⁵) and small-sized particles (F (2,13) = 24.30, P = 4.06 e⁻⁵) but not for the medium group (F (2,13) = 1.575, P = 0.244). f All AD and DS cases exhibited significant reductions (one-way ANOVA) in the large-to-small size ratios for particles from P2 fractions (F (2,13) = 29.83, P = 1.4 e⁻⁵). P values on bars are from two-sided Dunnett’s post hoc test comparing AD or DS vs control. g Plot showing the correlation between high-to-low immunoreactivity (IR) ratios for all FDT analyzed synapses (data for both PSD-95-ir and GPHN-ir in layers 1 and 2 were combined) and the large-to-small synaptosomal particles for each subject. Each subject is color coded by diagnosis: CTRL (gray), AD (cyan), DS (magenta). The solid line represents the linear fit; R²(16) = 0.72, P = 3.1 e⁻⁵, showing agreement between intensity and size data from FDT and flow cytometry driven by group separation.