Isolation and molecular profiling of primary mouse retinal ganglion cells

Comparison of phenotypes from healthy and glaucomatous retinas

Sumana R. Chintalapudi, Levon Djenderedjian, Andrew B. Stiemke, Jena J. Steinle, Monica Jablonski, Vanessa Morales-Tirado

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.

Original languageEnglish (US)
Article number93
JournalFrontiers in Aging Neuroscience
Volume8
Issue numberMAY
DOIs
StatePublished - Jan 1 2016

Fingerprint

Retinal Ganglion Cells
Retina
Phenotype
POU Domain Factors
Retinal Diseases
Retinal Degeneration
RNA-Binding Proteins
Homeobox Genes
Optic Nerve
Intraocular Pressure
Neurodegenerative Diseases
Visual Acuity
Flow Cytometry
Cell Culture Techniques
Brain
Population
Genes

All Science Journal Classification (ASJC) codes

  • Aging
  • Cognitive Neuroscience

Cite this

Isolation and molecular profiling of primary mouse retinal ganglion cells : Comparison of phenotypes from healthy and glaucomatous retinas. / Chintalapudi, Sumana R.; Djenderedjian, Levon; Stiemke, Andrew B.; Steinle, Jena J.; Jablonski, Monica; Morales-Tirado, Vanessa.

In: Frontiers in Aging Neuroscience, Vol. 8, No. MAY, 93, 01.01.2016.

Research output: Contribution to journalArticle

@article{5d5819069be64311abf5e9ad30c4a3e5,
title = "Isolation and molecular profiling of primary mouse retinal ganglion cells: Comparison of phenotypes from healthy and glaucomatous retinas",
abstract = "Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.",
author = "Chintalapudi, {Sumana R.} and Levon Djenderedjian and Stiemke, {Andrew B.} and Steinle, {Jena J.} and Monica Jablonski and Vanessa Morales-Tirado",
year = "2016",
month = "1",
day = "1",
doi = "10.3389/fnagi.2016.00093",
language = "English (US)",
volume = "8",
journal = "Frontiers in Aging Neuroscience",
issn = "1663-4365",
publisher = "Frontiers Research Foundation",
number = "MAY",

}

TY - JOUR

T1 - Isolation and molecular profiling of primary mouse retinal ganglion cells

T2 - Comparison of phenotypes from healthy and glaucomatous retinas

AU - Chintalapudi, Sumana R.

AU - Djenderedjian, Levon

AU - Stiemke, Andrew B.

AU - Steinle, Jena J.

AU - Jablonski, Monica

AU - Morales-Tirado, Vanessa

PY - 2016/1/1

Y1 - 2016/1/1

N2 - Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.

AB - Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases.

UR - http://www.scopus.com/inward/record.url?scp=84974559436&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84974559436&partnerID=8YFLogxK

U2 - 10.3389/fnagi.2016.00093

DO - 10.3389/fnagi.2016.00093

M3 - Article

VL - 8

JO - Frontiers in Aging Neuroscience

JF - Frontiers in Aging Neuroscience

SN - 1663-4365

IS - MAY

M1 - 93

ER -