June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Flow Cytometry Approach to Study NGF and p75 in Retinal Cells from Reeler mice
Author Affiliations & Notes
  • Bijorn Balzamino
    Lab Ophthalmology, IRCCS GB Bietti Foundation, Rome, Italy
  • Ramona Marino
    Laboratory of Developmental Neuroscience and Neural Plasticity, University Campus Bio-Medico, Rome, Italy
  • Graziana Esposito
    Lab Ophthalmology, IRCCS GB Bietti Foundation, Rome, Italy
  • Filippo Biamonte
    Institute of Histology and Embryology, Catholic University of the Sacred Heart “A. Gemelli”, Rome, Italy
  • Flavio Keller
    Laboratory of Developmental Neuroscience and Neural Plasticity, University Campus Bio-Medico, Rome, Italy
  • Alessandra Micera
    Lab Ophthalmology, IRCCS GB Bietti Foundation, Rome, Italy
  • Footnotes
    Commercial Relationships Bijorn Balzamino, None; Ramona Marino, None; Graziana Esposito, None; Filippo Biamonte, None; Flavio Keller, None; Alessandra Micera, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 6086. doi:
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      Bijorn Balzamino, Ramona Marino, Graziana Esposito, Filippo Biamonte, Flavio Keller, Alessandra Micera; Flow Cytometry Approach to Study NGF and p75 in Retinal Cells from Reeler mice. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6086.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Reeler mice appear to be a good model for exploring the cross-talk between NGF and Reelin in development/maintenance of a physiological retinal function. Therefore, we sought to characterize NGF and trkANGFR/p75NTR expression in retinal cells from Reeler-L7-EGFPreln-/- transgenic mice (E-Reeler, n=2) and C57BL/6J-L7-EGFPreln+/+ transgenic mice (E-control, n=7), both expressing EGFP positivity in Rod Bipolar Cells (RBC).

Methods: Retinas were dissected from not pooled whole eyes. Single cell were obtained by DispaseII and/or Trypsin digestion, in the presence of DNAseI, equilibrated in HBSS-EDTA and fixed in 1% PFA. Cells were probed with specific antibodies (NGF, p75NTR and trkANGFR) and at least 10000 cells were acquired/analysed by flow cytometry, according to the MACSquant technology. Apoptosis was also estimated by AnnexinV.

Results: Both treatments were successful to obtain single cells from dissected retinas, albeit trypsin allowed a better side-scatter definition/resolution of population. Three main cell populations were separated according to physical parameters: RBC/retinal ganglion cell (RGC), accessory cells (Muller, Amacrine, Horizontal) and photoreceptor. A slight expression of Annexin V was detected (2.9%). EGFP was detected only in RBC (9%). NGF and p75NTR were expressed mainly in RBC/RGC and faintly in accessories cells, while trkANGFR was weakly expressed by RBC and RGC. Both NGF and p75NTR were increased in GFP-positive RBC and RGC, from E-Reeler as compared to E-control.

Conclusions: From our studies, single cells can be enzymatic dissociated from unpooled retinas. The isolation method and the selective NGF/p75NTR increase in GFP-bearing RBC and RGC may provide a good experimental system for studying NGF-reelin cross-talk in these cells.

Keywords: 529 flow cytometry • 435 bipolar cells • 543 growth factors/growth factor receptors  
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