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. 2019 Nov 19;41(4):458–466. doi: 10.1093/carcin/bgz182

Figure 2.

Figure 2.

CD44+/CD271+ cells have higher growth rate in 2D and 3D culture conditions. (a) CD44+/CD271+, CD44+/CD271− and the unsorted parental cell population from SCC12 and SCC38 cell lines were seeded in 96-well plates and their cell growths were assessed using MTT assay (in triplicates from three independent experiments). Data are presented as mean ± SD (* p < 0.05 and ** p < 0.01 with CD44+/CD271−, #p < 0.05 and ##p < 0.01 with parental cells). (b) Assessment of self-renewal characteristic in 2D culture with the colony forming assay. Single cells from each of the three cell populations from SCC12 and SCC38 were seeded in 6-well plates and allowed to form colonies for 14 days (400 cells seeded per well). Fixed and stained colonies containing > 50 cells were counted under an inverted light microscope. Data are presented as mean ± SD (** p < 0.01). Sample photographs of the fixed and stained colonies are presented on the (c) panel. (d) To assess self-renewal in 3D culture, tumor-sphere formation assay was used. Single cells from each of the three cell populations from SCC12 were cultured in anchorage-independent and serum-free culture conditions and allowed to form spheres for 14 days. Data are presented as mean ± SD (** p < 0.01). (e) The spheres were dissociated into single cells and counted in the presences of trypan blue stain. Cells numbers are presented as mean ± SD (** p < 0.01). (f) The spheres were counted, and photos were taken under a phase-contrast microscope with ×5 (the main photos) and ×40 (the inserts) magnification. Scale bar = 90 µM and 10 µM, respectively.