Figure 6

CB ₂ stimulation modulates microglial phagocytic function. A, Mouse primary microglial cells were seeded in 6-well tissue culture plates with glass inserts (5 × 10⁵cells/well) and treated with 3 μM Cy3™-Aβ_1–42 in the absence (a and b; Control) or presence of either CD40L protein (c and d 2.5 μg/mL) or JWH-015 (e and f; 5 μM), or both JWH-015 and CD40L protein (g and h). After 3 hr these cells were washed and fixed (see Materials and Methods). Subsequently, immunofluorescence microscopy examination was performed using a 40 X objective with appropriate filter selection. The darkfield images a, c, e, and g show the fluorescence of Cy3™ labeled Aβ_1–42 whereas, b, d, f, and h show only the DAPI nuclear stain of the same fields. B, In parallel experiments, under the same treatment conditions, microglial cell lysates were prepared for Western immunoblotting analysis (see Materials and methods) of cell-associated Aβ_1–42 using anti-Aβ antibody (BAM-10, Sigma). C, Aβ mean band densities are graphically represented as ratios to β-actin +/- SD (n = 3 for each condition). ANOVA revealed significant between-group differences (JWH-015/Aβ versus CD40L/Aβ and Aβ/CD40L versus JWH-015/CD40L/Aβ; ** p < 0.005), and post hoc testing showed significant differences between CD40L/Aβ and JWH-015/CD40L/Aβ (** p < 0.005).