Figure 3.

PBM attenuates the neurotoxic effects of M1 macrophages on neurons. (A) Schematic diagram of the experiments with macrophages and neurons in pipette coculture. Timeline of the experiments conducted. (B) VSC 4.1 motor neurons were treated with M1 macrophage-conditioned medium (M1CM) for 24 hours, followed by double staining with Annexin V-FITC/PI and flow cytometry to detect apoptosis rates. Quadrant 3 (Q3) represents cells stained mainly by Annexin-V (early apoptotic cells), and Q2 represents cells stained by both PI and Annexin-V (late apoptotic). Q1 represents cells stained mainly by PI, and viable cells negative for both Annexin-V and PI appear in Q4. (C) The apoptosis rate of neuronal cells is shown in the bar graph. (D) Representative images of β-III-tubulin⁺ cultured DRG neurons (n=3 independent experiments for neurite outgrowth assays, scale bars=50 μm). (E) A quantification graph of the mean neurite length per neuron in each group. (F) ELISA was used to detect the concentration of inflammation-associated cytokines in macrophage-conditioned culture media (all experiments in this group were independently repeated three times). *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant.