大量纯化的视网膜神经节细胞（RGCs）作为体外研究的工具，在研究神经再生和治疗青光眼的治疗方法的发展方面具有重要价值。传统上，RGCs是从早期出生的大鼠和小鼠中分离得到的，最近也从人体体外培养的视网膜器官样体中，利用一种基于Thy-1表达的两步免疫粘附技术进行分离。然而，这种技术限制了可以分离RGCs的时间段，错过了最早出生的RGCs，这是轴突生长最活跃的阶段，同时，在视网膜退行性疾病模型中使用有限，因为受伤后Thy-1会下调。尽管荧光相关细胞分选（FACS）结合新的光遗传标记的RGCs可以克服这个限制，但传统FACS分选器的使用仅限于基因组学和蛋白质组学研究，因为RGCs在分选后几乎没有存活。在这里，我们描述了一种新的RGC分离方法，利用了联合免疫粘附-荧光相关细胞分选（IP-FACS）协议，在使用低压FACS分离这些细胞之前，通过免疫粘附法来减少巨噬细胞和光感受器。我们证明，通过IP-FACS分离得到的RGCs与相同年龄通过免疫粘附法分离得到的RGCs相比，其抗体染色和qRT-PCR测定的纯度、活存率、神经突起生长和钙释放反应到谷氨酸的电生理性能具有类似性。最后，我们证明了在Thy-1表达之前可从早期胚胎小鼠中分离得到RGCs的能力，使用Brn3b-eGFP的光遗传标记细胞。该方法提供了一种用于研究早期发育的RGCs、RGC亚型研究和RGC细胞移植研究的新方法。版权所有 © 2023 Mcloughlin，Aladdad，Payne，Boda，Nieto-Gomez和Kador。

Purified Retinal Ganglion Cells (RGCs) for in vitro study have been a valuable tool in the study of neural regeneration and in the development of therapies to treat glaucoma. Traditionally, RGCs have been isolated from early postnatal rats and mice, and more recently from human in vitro derived retinal organoids using a two-step immunopanning technique based upon the expression of Thy-1. This technique, however, limits the time periods from which RGCs can be isolated, missing the earliest born RGCs at which time the greatest stage of axon growth occurs, as well as being limited in its use with models of retinal degeneration as Thy-1 is downregulated following injury. While fluorescence associated cell sorting (FACS) in combination with new optogenetically labeled RGCs would be able to overcome this limitation, the use of traditional FACS sorters has been limited to genomic and proteomic studies, as RGCs have little to no survival post-sorting. Here we describe a new method for RGC isolation utilizing a combined immunopanning-fluorescence associated cell sorting (IP-FACS) protocol that initially depletes macrophages and photoreceptors, using immunopanning to enrich for RGCs before using low-pressure FACS to isolate these cells. We demonstrate that RGCs isolated via IP-FACS when compared to RGCs isolated via immunopanning at the same age have similar purity as measured by antibody staining and qRT-PCR; survival as measured by live dead staining; neurite outgrowth; and electrophysiological properties as measured by calcium release response to glutamate. Finally, we demonstrate the ability to isolate RGCs from early embryonic mice prior to the expression of Thy-1 using Brn3b-eGFP optogenetically labeled cells. This method provides a new approach for the isolation of RGCs for the study of early developed RGCs, the study of RGC subtypes and the isolation of RGCs for cell transplantation studies.Copyright © 2023 Mcloughlin, Aladdad, Payne, Boda, Nieto-Gomez and Kador.