Purification and separation of retinal ganglion cells by retrograde fluorescence labeling and fluorescent activated cell sorting (FACS)

Abstract

Abstract: : Purpose:The knowledge of different cell types and their interaction is necessary for the understanding of regeneration, degeneration and the reaction to toxic stimuli. Therefore separation of different cell types and the collection of purified cells are required for further experimental studies. An established method for retrograde fluorescence labeling of retinal ganglion cells (RGC) is combined with the fluorescent activated cell sorting (FACS) method to seperate and enrich RGC within cell suspensions. Methods: RGC were retrograde labeled for the specific requirements of the FACS system with the tracer Di–Asp. The excitation and emission spectrums are at 492nm and 590 nm and exactly within the detection range of the used FL2–detector of the FACS machine. After injection of the tracer in the projection area of RGC in rats, the dye was taken up by the axon terminals and transported to the contralateral soma of the RGC within 48 hours. Positive fluorescence in RGC is therefore a morphological correlate of intact axonal transport of RGC. Retrograde marked adult or neonatal rats (age P 7–12) were decapitated and eyes enucleated. Isolated retinae can enzymatically digested for 12 minutes in D/L–cystein activated papain–solution (4.5 units/ml). Remained cell–clusters are pipetted in a 1ml glass–pipette for approximately 30 times. Afterwards single RGC can be identified under fluorescence microscopy in the suspension. Results: Identification of fluorescence labeled RGC was performed by a FACSCaliburTM, Becton Dickinson Co. Different tracers are used to obtain maximal results in sorting marked RGC and tested for biological compatibility. Separation of fluorescent labeled cells was carried out by the FACSortTM system. The sorting can be done in a sterile system and allows therefore cultivation of separated cells for further analysis. By using a cell concentrator, cells can be directly sorted to sterile filters or sterile cell culture inserts. Beside the separation of retrograde labeled RGC from other retinal cells, the identification of different subtypes of RGC using immunostaining is possible. Conclusions: The collected cells can be used for further analysis using specific markers for surface antigens. For different pathologies like trauma or glaucoma like conditions cells can be analyzed for cell size and purified cell suspensions can be obtained for possibly further protein analysis.

Keywords: ganglion cells • detection • imaging/image analysis: non–clinical