Quantification of retinal pigment epithelial phenotypic variation using laser scanning cytometry

[摘要] Purpose: Quantifying phenotypicvariation at the level of protein expression (variegation) withinpopulations of retinal pigment epithelium (RPE) cells may be importantin the study of pathologies associated with this variation. The lack ofquantitative methods for examining single cells, however, and thevariable presence of pigment and/or lipofuscin complicate thisexperimental goal. We have applied the technique of laser scanningcytometry (LSC) to paraffin sections of mouse and human eyes toevaluate the utility of LSC for these measurements. Methods: Mouse eyes were perfusion fixedin 4% paraformaldehyde and embedded in paraffin. Postmortem human eyeswere fixed and dissected to obtain a 9-mm punch, which was thenembedded in paraffin. A laser scanning cytometer equipped with violet,argon, and helium-neon lasers and the detectors for blue, green, andlong red were used to record the fluorescence of each individual cellat all three wavelengths. Raw data were recorded and processed usingthe WinCyte software. Individual nuclei were identified by thefluorescence of the 4’,6-diamidino-2-phenylindole (DAPI) nuclearcounterstain. Next, RPE cells were uniquely identified in the greenchannel using an anti-retinal pigment epithelium-specific protein65 kDa (anti-RPE65) monoclonal antibody with an Alexa Fluor488-labeled secondary antibody. Mn-superoxide dismutase (MnSOD) wasquantified in the long-red channel using an anti-MnSOD antibody and anAlexa Fluor 647-labeled secondary antibody. MnSOD+ and RPE65+cells exhibited peaks in the plot of fluorescence intensity versus cellnumber, which could be characterized by the mean fluorescence intensity(MFI), the coefficient of variation (CV), and the percentage of totalRPE cells that were also labeled for MnSOD. Results: RPE cells can be uniquelyidentified in human and mouse paraffin sections by immunolabeling withanti-RPE65 antibody. A second antigen, such as MnSOD, can then beprobed only within this set of RPE. Results are plotted primarily withthe population frequency diagram, which can be subdivided into multipleregions. The data collected for each region include the MFI, the CV,and the number of cells that are immunolabeled in that region.Background interference from pigment or autofluorescent material can besuccessfully overcome by elevating the concentrations of fluorescentsecondary antibodies. In the human and mouse eyes, age-related changesin MFI, CV, and percent RPE cells immunolabeled for MnSOD wereobserved. Conclusions: The extent of thevariability of gene expression in RPE cells at the protein level can bequantified by LSC. Relative changes in the MFI, the CV, and/orpercentage of RPE cells double labeled for a second antigen quantifythe changes observed. The analysis of these data also suggest whetherthe effects observed are related to local changes in transcription(alterations of CV) or major changes of protein expression (MFI), whichare likely to be due to changes in the chromatin structure. The changesof these variables with age suggest that the observed age-relatedvariegation is primarily due to changes in the chromatin structure inindividual cells.

[学科分类] 生物化学/生物物理