Quantification of neuronal cells in healthy rat retinas by flow cytometry

Abstract

Abstract Purpose 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. Methods Rats were killed and the eyes were enuclated to achieve retinal dissection. Tissue dissociation was accomplished with trypsin. After trypsin action was blocked the cells were mechanically dissociated into a single‐cell suspension by gentle pippeting. The 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 100.000 healthy cells were analyzed with a FACScalibur and FlowJo software. Dead cell and cell debris were exluded from analysis by gating with forward scatter and side scatter as indicators. The primary antibodies were anti‐rhodopsin against photoreceptors, anti‐Protein Kinace C against bipolar cells, anti‐calbindin against horizontal cells and anti‐microtubule associated protein 1 against ganglion cells. All the primaries antibodies were monoclonal and the secondary antibody was goat anti‐mouse Alexa Fluor 594. Results Quantification of the above populations was possible using flow cytometry. In this preliminary study, the photoreceptors had the 53.99%, the ganglion the 7%, the bipolars the 4% and the horizontal the 1.15% in the hole mixed retinal population. All experiments were repeated six times. Conclusion Flow cytometry can be used to quantify the different neuronal populations in control healthy eyes and this verification will be very useful in future in studies in apoptosis or proliferation of these cells.