Redvers et al. 10.1073/pnas.0602579103. |
Supporting Figure 6
Supporting Materials and Methods
Fig. 6. Epidermal side population (SP) lineage analysis. Expression of K14 was examined in SP cytospins by immunohistochemistry (a) and in freshly isolated Hoechst subsets by FACS (b) to assess the frequency and differentiation status of keratinocytes. Unfractionated (Unf) cells were stained with Hoechst, followed by the melanocyte marker c-kit (c and d; n = 3) or Langerhans cell/intraepithelial lymphocyte marker CD45 (e and f; n = 4). A rare subset of total viable cells were c-kit+ (c), and, when the Hoechst profile of these cells was overlayed on that of the total sample, none were found in the SP region (d). Similarly, an infrequent subset of CD45+ cells (e) were all located in the non-side population (NSP) region (f). Oil red O staining on cytospins of sorted cells demonstrated rare sebocytes in Unf cells (g) but did not identify any in the SP fraction (h).
Supporting Materials and Methods
Phenotypic Characterization of Hoechst Fractions. K14 was detected in 100% methanol-fixed sorted cells with mAb LL001 (1:200; Irene Leigh, University of London, U.K.) and the VECTASTAIN ABC peroxidase kit with AEC substrate (Vector Laboratories, Burlingame, CA) or anti-mouse IgG-FITC (1:80, Jackson ImmunoResearch, West Grove, PA). Hoechst-stained sorted epidermal cells were immunostained for c-kit (ACK45, 1:100) and CD45 (30-F11, 1:100) with antibodies from BD Biosciences (San Jose, CA) as described (1). Oil red O staining was performed with a 0.3% (w/v) solution in 60% isopropanol for 10 min at room temperature.
1. Li, A. & Kaur, P. (2005) in Epidermal Cells: Methods and Protocols, ed. Turksen, K. (Humana, Totowa, NJ), Vol. 289, pp. 87–96.