Figure 4
Purification of human SC cultures via MACS. Results from representative experiments using unpurified human SC populations containing high (a) and low (b–c) fibroblast contamination are shown. Cells were labeled in suspension with p75NGFR antibodies and separated using MS-columns (Methods). Samples from the pre-sorting (total) and post-sorting cells were plated in SC growth medium (retained cells) and FBS-only medium (eluted cells) for analysis by immunofluorescence microscopy and flow cytometry. The retained and eluted cells were highly viable (c), rapidly adhered to the substrate acquiring the expected pattern of growth (a–c) and underwent proliferation, as denoted by EdU labeling (red nuclei, c). Representative images of a senescent human SC culture (passage-4) subjected to MACS is shown in panel c (bottom images, SA-βGAL activity) to confirm the absence of senescent cells in the eluted fraction. Observe the negligible representation of S100β and p75NGFR positive cells in the populations of eluted cells (a–c) and the high SC enrichment obtained in one round of purification (a). A quantification of the proportion of proliferating (EdU positive) cells discriminated on the basis of p75NGFR expression (green) is shown in panel (d) along with an assessment of viability (Trypan blue exclusion assays) for cells in suspension prior to (total) and immediately after MACS (n = 8).
