We further examined neurite length by the co-culture method

ration was monitored for 24 hours in the presence of LPS and expressed as the increase in total cellular protein in either control medium (25 mM glucose) or medium containing 2.5 mM oligomycin and 25 mM glucose (A), 10 mM 2-DG and 25 mM glucose (B), or 10 mM galactose and no glucose (C). Cell viability was also assessed for 24 hours LY-411575 web pubmed ID:http://www.ncbi.nlm.nih.gov/pubmed/19652232 under the same medium conditions in the presence of pSIVA apoptosis biosensor [50] (D). The appearance of the fluorescent pSIVA signal was recorded in real time and the total pixel area per frame was measured using Fiji Imaging software. An increase in the pSIVA pixel area correlates linearly with increase in the amount of apoptotic cells. Data in A�C represent means 6 SEM of three independent experiments performed in triplicate and in D the averages of one experiment performed in triplicate. (p,0.05, p,0.01, p,0.001, unpaired t-test). doi:10.1371/journal.pone.0096786.g001 microscopy of (phalloidin-stained) RAW 264.7 cells before and after LPS-stimulation. Oligomycin treated cells preserved the ability to spread upon LPS-stimulation and appeared to have more filopodia than control cells (Figure 3A�F and Figure 4A�F). These thorn-like protrusions became clearly visible after 6 hours (not shown) and were observed on both LPS stimulated and unstimulated cells. Quantification of the number of filopodia extending radially from the cell body after 24 hours, confirmed that oligomycin induced filopodia formation in RAW 264.7 cells (Figure 3K and 4K) although the extent of oligomycin effects (on cells with and without LPS) as determined by fluorescence and SEM microscopy varied. Partly this variation may be due to differential effects of the fixation-staining, and also to the semiquantitative nature of