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.

Balzamino, B. O., Marino, R., Esposito, G., Biamonte, F., Keller, F., Micera, A., Flow Cytometry Approach to Study NGF and p75 in Retinal Cells from Reeler mice, Abstract de <<ARVO 2013>>, (Seattle, 28-28 October 2013 ), Arvo, Seattle 2013: N/A-N/A [http://hdl.handle.net/10807/62087]

Flow Cytometry Approach to Study NGF and p75 in Retinal Cells from Reeler mice

Biamonte, Filippo;
2013

Abstract

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.
eng
ARVO 2013 Annual Meeting
ARVO 2013
Seattle
28-ott-2013
28-ott-2013
E- 6086
Balzamino, B. O., Marino, R., Esposito, G., Biamonte, F., Keller, F., Micera, A., Flow Cytometry Approach to Study NGF and p75 in Retinal Cells from Reeler mice, Abstract de <>, (Seattle, 28-28 October 2013 ), Arvo, Seattle 2013: N/A-N/A [http://hdl.handle.net/10807/62087]
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10807/62087
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