Skeletal Muscle Neural Precursor Figure 9. Nestin-GFP+ cell sorting and culture. A. Representative scatter plots showing fluorescence-activated sorting of cells derived from nestin-GFP+ hindlimb muscles immediately after isolation. GFP fluorescence was plotted against the number of cells. We selected cells with very high (P2) and no GFP fluorescence (P4). Sorts of total events yielded 1.9% GFP+. Histograms of re-analysis of sorted GFP- (B) and GFP+ cells (C). Note that B shows some GFP- cells, which probably result from a decline in fluorescence with time. D. Percent of nestin-GFP+ cells before and after FACS. E. Number of nestin-GFP+ cells differentiated into neurons after culture in different conditions: nestin-GFP- or nestin-GFP+ cells cultured alone; co- cultured nestin-GFP+ and nestin-GFP- cells; nestin-GFP- or nestin-GFP+ cells co-cultured with wild-type FDB fibers; and nestin-GFP+ or nestin-GFP- cells co-cultured with medium from wild-type mouse FDB analyzed after 8 days in culture. The number of nestin-GFP+ and nestin-GFP- plated cells were approximately 5000 and 40,000 per dish, respectively, in all conditions. Only GFP+ cells with 2 or more processes were counted. Data are mean 6 SEM. doi:10.1371/journal.pone.0016816.g009 PLoS ONE | www.plosone.org 13 February 2011 | Volume 6 | Issue 2 | e16816