Separation of FRCC by flow cytometry. (A) A representative flow cytometry plot of FRCC separated according to light scatter characteristics reflecting cytoplasmic granularity and cell size. Seven independent replicates of the sorting procedure were used to isolate the S (lowest 15% FSC and lowest 25% SSC) and L (highest 15% FSC and highest 25% SSC) subpopulations. This cytogram shows debris close to the origin but particles smaller than 5 μm were excluded from the sort. (B) Histogram showing the number of sorted cells (▪; mean ± SEM) in each population that attached to glass after overnight plating compared to replated FRCC from the same cultures (▨). Counts were done on DAPI-stained cells and expressed as number per 100 cells plated. (C) Analysis of OPN mRNA expression in sorted cells measured by RT-PCR. Lanes 1 through 5, S cells; FRCC; L cells; S⁻ cells; L⁻ cells. First, amplification of an 871-bp fragment, encompassing most of the OPN sequence, was performed using total RNA extracted from the cells (left side). This was followed by amplification of a 486-bp fragment using the 871 fragment as template (right side), which confirmed OPN expression in all populations except the S population.