Isolation of primary mouse retinal ganglion cells using immunopanning-magnetic separation

Samin Hong, Yoko Iizuka, chanyun kim, Gong Je Seong

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

Purpose: To establish an effective system for isolating primary retinal ganglion cells (RGCs) from newborn mice. Methods: The retinas were separated from enucleated eyeballs of Crl:CD-1 mice on postnatal day 1 to 4. RGCs were purified using three different methods, including two-step immunopanning (TSI), direct magnetic separation (DMS), and immunopanning-magnetic separation (IMS). Harvested cells were maintained for 24 h in a defined medium and then examined with immunocytochemistry, western immunoblotting, and real-time reverse transcription polymerase chain reaction (RT-PCR) for glial cell-specific glial fibrillary acidic protein (GFAP) and amacrine cell-specific syntaxin 1. Results: As determined with immunofluorescence staining, RGCs purified by TSI were sparsely mixed with GFAP-positive astrocytes, and RGCs isolated by DMS were frequently mixed with syntaxin 1-positive amacrine cells. However, RGCs collected by IMS were seldom contaminated by GFAP-positive or syntaxin 1-positive cells. On western immunoblots, TSI cells showed significant GFAP expression, and DMS cells showed apparent syntaxin 1 expression, but IMS cells did not. Results of the real-time RT-PCR showed a similar tendency to those of the immunocytochemistry and western immunoblots. Conclusion: Primary mouse RGCs were highly purified by the IMS method, combining the benefits of the TSI and DMS methods. This isolation method may provide a good experimental system for studying glaucoma in vitro.

Original languageEnglish
Pages (from-to)2922-2930
Number of pages9
JournalMolecular Vision
Volume18
Publication statusPublished - 2012 Dec 3

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Retinal Ganglion Cells
Syntaxin 1
Glial Fibrillary Acidic Protein
Amacrine Cells
Cell Separation
Western Blotting
Reverse Transcription
Immunohistochemistry
Polymerase Chain Reaction
Neuroglia
Astrocytes
Glaucoma
Fluorescent Antibody Technique
Retina
Staining and Labeling

All Science Journal Classification (ASJC) codes

  • Ophthalmology

Cite this

Hong, Samin ; Iizuka, Yoko ; kim, chanyun ; Seong, Gong Je. / Isolation of primary mouse retinal ganglion cells using immunopanning-magnetic separation. In: Molecular Vision. 2012 ; Vol. 18. pp. 2922-2930.
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Isolation of primary mouse retinal ganglion cells using immunopanning-magnetic separation. / Hong, Samin; Iizuka, Yoko; kim, chanyun; Seong, Gong Je.

In: Molecular Vision, Vol. 18, 03.12.2012, p. 2922-2930.

Research output: Contribution to journalArticle

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