Abstract
Abstract: :
Purpose: Loss of retinal ganglion cells via the cell death process apoptosis is a key feature of glaucoma. We developed an alternative approach to the two-step panning method for the isolation of retinal ganglion cells. In this report we describe a novel procedure based on flow cytometry for the identification of a specific subset of cells in a mixed cell population. This assay is based on the expression of Thy-1, a cell-type-specific marker. This subset of retinal ganglion cells was used to investigate the regulation of ganglion cell apoptosis. Methods: Adult Wistar or Sprague-Dawley rats were used in all experiments. Enucleated eyes were placed in HBSS. The retina was dissected and placed in cold medium. Tissue dissociation was achieved in a 0.25% Trypsin containing Dnase II and incubated @ 37°c for 17'. Dissociation was completed by trituration of trypsin treated tissue with a pasteur pipette. Retinal cells were fixed in 1% formaldehyde @ RT for 30' then washed in PBS containing 0.1M glycine. Cells were resuspended in mouse monoclonal Thy-1 antibody (diluted in 1% BSA in PBS) and incubated on ice for at least 2h. Cells were then incubated on ice for 30’ with FITC-conjugated anti-mouse IgG. Samples were read on a FACScan flow cytometer equipped with CELLQuest software. FITC fluorescence was collected through FL1-H channel and plotted against forward light scatter (FSC-H). A minimum of 10,000 events was collected for each sample. Results: Thy-1 antibody allowed the identification of a discrete population of retinal ganglion cells which represent a small proportion (approximately 1.5%) of the total cell population. Thy-1 positive retinal ganglion cells showed increased FL1-H fluorescence due to binding of the FITC-Conjugated secondary antibody to the bound Thy-1 antibody. These cells were used to investigate the regulation of apoptosis and to this end were incubated with physiologically relevant stimuli such as L-glutamate and its agonist NMDA. Conclusion: The results summarised above indicate that the method of isolation and fixation of retinal cells implemented maintains the integrity of the Thy-1 epitope, which validates the use of this method for the isolation and detection of retinal ganglion cells. The ability to identify the retinal ganglion cell population in this way is of great benefit since flow cytometry not only facilitates the detection and measurement of apoptosis itself but also permits the detection of intracellular changes such as the production of ROS, mitochondrial depolarisation, calcium concentration, and plasma membrane asymmetry.
Keywords: apoptosis/cell death • ganglion cells • flow cytometry