Comparative analysis of three purification protocols for retinal ganglion cells from rat

PURPOSE To make comparative analyses of the common three purification protocols for retinal ganglion cells (RGCs), providing a solid practical basis for selecting the method for purifying RGCs for use in subsequent experiments. METHODS Rat RGCs were isolated and purified using three methods, including two-step immunopanning (TIP) separation, two-step immunopanning-magnetic (TIPM) separation, and flow cytometric (FC) separation. Immunocytochemical staining, quantitative real-time PCR, flow cytometry, electrophysiology, and Cell Counting Kit-8 (CCK-8) analyses were performed to compare the purity, yield, and viability of the RGCs. RESULTS The RGC yields from the TIP, TIPM, and FC methods were 24.60±15.98 × 10(4), 5.28±4.42 × 10(4), and 5.4±2.7 × 10(3) per retina, respectively. We easily controlled the relative purity of the RGCs with the FC method and even reached 100% of the maximum expected purity. However, the RGC purity was only 80.97±5.45% and 95.41±3.23% using the TIP and TIPM methods, respectively. The contaminant cells were mainly large, star-shaped, glial fibrillary acidic protein (GFAP)-positive astrocytes and small, round, syntaxin 1-positive amacrine cells with multiple short neurites. The RGCs purified with FC could not be cultured successively in our study; however, the TIP-RGCs survived more than 20 days with good viability, while the TIPM-RGCs survived less than 9 days. CONCLUSIONS The three protocols for purifying the RGCs each had its own pros and cons. The RGCs isolated by the TIP method exhibited the highest viability and yield but had low purity. The purity of the RGCs isolated with the FC method could reach approximately 100% but had a low yield and cell viability. The TIPM method was reliable and produced RGCs with considerable purity, yield, and viability. This study provides a solid practical basis for selecting the method for purifying RGCs for use in subsequent experiments.