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Figure 2. Characteristics of different cell sizes as determined by FACS analysis. (A) Representative FACS dot plot of cultured oral keratinocytes at passages 2 and 4. Shown are the 3 gates (Small, Medium, Large) for sorting cells by relative cell size in diameters in equal proportions (approximately 25%) (left panels). The subpopulation indicated by a black arrow in the dot diagrams was debris eliminated by the gate in the side-scatter vs. forward-scatter, as well as PI-fluorescence before cell-sorting. (right panels) ß1 integrin expression at passage 2 in each size of sorted cells. ß1 integrin was expressed 99.4 ± 0.6% in Large cells, 99.5 ± 0.4% in Medium cells, and 99.4 ± 0.5% in Small cells. (B) Representative PPAR
expression at passages 4 (72.8% in Large cells, 57.3% in Medium cells, 44.0% in Small cells) and 6 (84.2% in Large cells, 83.9% in Medium cells, 49.5% in Small cells) in each size of sorted cells. Among the cell size groups, the different proportion of PPAR-positive cells was statistically significant (79.9 ± 25.5% in Large cells, 57.6 ± 34.7% in Medium cells, 37.4 ± 32.6% in Small cells) (overall p < 0.0001). Dotted line drawn at the top 3% of the total population of background control indicates the borderline showing positive FITC fluorescence when compared with background control. Note shift of the histograms to the right as the passage number increases, indicating that a larger percentage of cells is PPAR-positive. (C) Linear regression analysis with a linear mixed model, showing relationship between proportion cells in S + G2M phases and days in culture in each size group. Passage numbers ranged from 1 to 8. The longer Small cells were in culture, the more cycling cells increased. In contrast, the longer Medium and Large cells were in culture, the more non-cycling cells increased. (D) Linear regression analysis with a linear mixed model, showing relationship between proportions of cells in S + G2M phases and cell diameters in each size group. Passage numbers ranged from 1 to 8. Within Small cells, the smaller they were, the more quiescent. In contrast, within Medium and Large cells, the smaller they were, the more cycling occurred.
