387 685

Cited 0 times in

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

Authors
 Samin Hong  ;  Yoko Iizuka  ;  Chan Yun Kim  ;  Gong Je Seong 
Citation
 MOLECULAR VISION, Vol.18 : 2922-2930, 2012 
Journal Title
MOLECULAR VISION
Issue Date
2012
MeSH
Amacrine Cells/cytology ; Amacrine Cells/metabolism ; Animals ; Animals, Newborn ; Antibodies/chemistry ; Antibodies/immunology ; Astrocytes/cytology ; Astrocytes/metabolism ; Biomarkers/metabolism ; Blotting, Western ; Female ; Gene Expression ; Glial Fibrillary Acidic Protein ; Immunohistochemistry ; Immunomagnetic Separation/methods* ; Immunomagnetic Separation/standards ; Mice ; Nerve Tissue Proteins/genetics ; Nerve Tissue Proteins/metabolism ; Primary Cell Culture ; Real-Time Polymerase Chain Reaction ; Retinal Ganglion Cells/cytology* ; Retinal Ganglion Cells/metabolism ; Syntaxin 1/genetics ; Syntaxin 1/metabolism
Keywords
Amacrine Cells/cytology ; Amacrine Cells/metabolism ; Animals ; Animals, Newborn ; Antibodies/chemistry ; Antibodies/immunology ; Astrocytes/cytology ; Astrocytes/metabolism ; Biomarkers/metabolism ; Blotting, Western ; Female ; Gene Expression ; Glial Fibrillary Acidic Protein ; Immunohistochemistry ; Immunomagnetic Separation/methods* ; Immunomagnetic Separation/standards ; Mice ; Nerve Tissue Proteins/genetics ; Nerve Tissue Proteins/metabolism ; Primary Cell Culture ; Real-Time Polymerase Chain Reaction ; Retinal Ganglion Cells/cytology* ; Retinal Ganglion Cells/metabolism ; Syntaxin 1/genetics ; Syntaxin 1/metabolism
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.
Files in This Item:
T201204100.pdf Download
Appears in Collections:
1. College of Medicine (의과대학) > Dept. of Ophthalmology (안과학교실) > 1. Journal Papers
1. College of Medicine (의과대학) > Yonsei Biomedical Research Center (연세의생명연구원) > 1. Journal Papers
Yonsei Authors
Kim, Chan Yun(김찬윤) ORCID logo https://orcid.org/0000-0002-8373-9999
Seong, Gong Je(성공제) ORCID logo https://orcid.org/0000-0002-5456-4296
Iizuka, Yoko(이주카요코)
Hong, Sa Min(홍사민)
URI
https://ir.ymlib.yonsei.ac.kr/handle/22282913/91480
사서에게 알리기
  feedback

qrcode

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.

Browse

Links