Fig. 2

Mixed cultures of neurons, oligodendrocytes, and astrocytes demonstrate reduced complement cytotoxicity. Neuroglial mixed cultures prepared from PLP-eGFP mouse pups were treated with 5% HC for 4 h in the presence of DRAQ7 and imaged using IncuCyte. a Phase contrast images of IncuCyte cultures treated with human complement (HC) at 0 and 4 h. Insert: Fluorescence image showing oligodendrocytes (PLP-eGFP; green) and dead cells (DRAQ7; red). Arrows: neurons. Arrow heads: astrocytes. b Quantitation of the percentage of DRAQ7+ neurons at 2 and 4 h in the presence of 2% or 5% HC. c Fluorescence image and quantitation of the percentage of PI+ dead neurons following exposure to 5% HC for 4 h. Left: fluorescence image of mixed cultures exposed to CTRL or HC and stained with PI (red) followed by NeuN (green) at 4 h. Note the dead neurons (PI+/NeuN+) in the HC-treated cultures. d, e IncuCyte fluorescence images (left) and quantitation of (d) differentiated oligodendrocyte (OL, arrows) and (e) oligodendrocyte progenitor (OPC) death in mixed cultures. OPCs and differentiated OLs were defined by morphology. More than 150 differentiated oligodendrocytes (OL) were counted for each experiment. CTRL: PBS. Scale bars 100 μm