Figure 5. Separation of SCs and fibroblasts by MACS after cell surface immunolabeling with Thy-1 or p75^NGFR antibodies.

(a–h) MACS separation of Thy-1-labeled cells from acutely isolated adult (a–e) and postnatal (f–h) rat nerve-derived cultures. Labeling and purification was performed as described in Methods. Cells were analyzed by immunofluorescence microscopy prior to (total fraction) and after MACS separation using the markers indicated in the figure (a,c,d,f,h). The retained and eluted fractions from each purification round were collected and analyzed in parallel. Low magnification images of cultures stained with Thy-1 (a,f) are shown along with a quantification of the results (b,g, bars represent mean ± SD, ***p < 0.001). Note the complete elimination of fibroblasts in preparations from both adult and postnatal sciatic nerves. An estimation of the percentage of remaining fibroblasts in the eluted fractions from adult and postnatal cells determined that only 0.3 ± 0.2% and 0.8 ± 1.0% of the cells, respectively, expressed Thy-1 immunoreactivity. Prior to purification, samples from adult and postnatal cells contained 21.11 ± 4.35% (b) and 14.83 ± 2.56% (g) of Thy-1 positive cells, respectively. In (e), duplicate samples from independent culture wells were analyzed by Western blot using antibodies against Thy-1, GFAP,CNPase, nestin and MCM2 (proliferation marker). Expression of β-actin served for reference control and confirmation of equal protein loading. (i–k) MACS separation of p75^NGFR-labeled cells from acutely isolated adult nerve cultures (a–e). Immunofluorescence image analysis of the total and retained fractions confirmed the effectiveness of separation (i). A quantification analysis of the cells present in the retained fraction confirmed that the percentage of Thy-1 positive cells was as low as 0.1 ± 0.1% (j) and that the remaining of the cells (98.5 ± 0.8%) were identified as p75^NGFR positive SCs (k) (bars = mean ± SD, ***p < 0.001).