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L. M. Hjelmeland, A. Fujikawa, S. L. Oltjen, Z. Smit-McBride, D. Braunschweig; Quantification of RPE Gene Expression by Laser Scanning Cytometry. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2935.
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The examination of RPE gene expression at the mRNA and protein levels can lead to apparent mosaicism or variegation. The lack of quantitative methods for examining single cells, however, and the variable presence of pigment and/or lipofuscin complicates the interpretation of immunofluorescence images. We have applied the technique of Laser Scanning Cytometry (LSC) to paraffin sections of mouse and human eyes in order to assess whether multiple populations of RPE cells exist with different levels of gene expression.
Mouse eyes were perfusion fixed in 4% paraformaldehyde and embedded in paraffin. Macular punches (9 mm) were prepared from human globes obtained from FFB. A laser scanning cytometer equipped with violet, argon and HeNe lasers and the following detectors: blue (463/DF50), green (530/30BP) and long red (650 EFLP) was used to record the fluorescence of each individual cell at all three wavelengths. Raw data were recorded and processed using WinCyte software provided by the LSC manufacturer (CompuCyte Corporation, Cambridge, MA).
Individual cells were localized by the fluorescence of the DAPI nuclear counterstain in the blue channel. Doublets and debris were removed by gating on the single cell population of a Blue Integral (total blue fluorescence within a cellular contour) vs. Area scattergram. RPE cells were then uniquely identified using RPE65 fluorescence in the green channel. The expression of a second antigen or cRNA probe was then quantified within the set of RPE cells using the long red channel.Several antigens, which visually appeared to exhibit mosaic or variegated expression, showed only one peak when the data were plotted as cell number vs. fluorescence intensity.
Our interpretation of these data suggest a single population of RPE cells with a wide variability of gene expression for individual antigens as indicated by large values of the coefficient of variance (CV) (broad peaks) in the cell number vs. fluorescence intensity diagrams.
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