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P. A. Tsoka, E. Koutala, E. A. Papadaki, I. G. Pallikaris, M. K. Tsilimbaris; Quantification of Neuronal Cells in Healthy Rats' Retinas by Flow Cytometry. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2149.
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The primary purpose of this study was to evaluate the potential to quantify the different neuronal populations in the retina of healthy Sprague-Dawley rats in an accurate quantitative way by using flow cytometry.
Rats (250-300 gr) were killed and the eyes were enuclated to achieve retinal dissection. Tissue dissociation was accomplished with trypsin. Trypsin action was blocked with cell medium containing FBS and gentamycin. The cells then were mechanically dissociated into a single-cell suspension by gentle pippeting. The dissociated cells were permeabilized and stained with the primary antibody. This incubation was followed by another one with a blocking antibody and finally the cells were incubated with the secondary antibody. At least 40.000 healthy cells were analyzed with a FACScalibur and FlowJo software. Dead cell and cell debris were exluded from analysis by gating with forward scatter (FCS, cell size) and side scatter (SSC, cell complexity) as indicators. The primary antibodies were anti-rhodopsin against photoreceptors, anti-Protein Kinace C against bipolar cells, anti-calbindin against horizontal cells, anti-choline acetyltranferace against cholinergic amacrine cells and anti-microtubule associated protein 1 against ganglion cells. All primary antibodies were monoclonal and the secondary antibody was goat anti-mouse Alexa Fluor 594.
Quantification of the above populations was possible using flow cytometry. In this preliminary study, the photoreceptors had the 53.99% (Figure), the ganglion the 7.34%, the bipolars the 4.04%, the horizontal the 3.5% and the cholinergic amacrine cells the 1.23% in the hole mixed retinal population. The experiments were repeated three times and these measurements are the mean value for each cell category.
Flow cytometry can be used to quantify the different neuronal populations in control healthy eyes and this verification can be very useful in future studies of apoptosis or proliferation of these cells.
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