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  • The mito-QC Reporter for Quantitative Mitophagy Assessment in Primary Retinal Ganglion Cells and Experimental Glaucoma Models.

The mito-QC Reporter for Quantitative Mitophagy Assessment in Primary Retinal Ganglion Cells and Experimental Glaucoma Models.

International journal of molecular sciences (2020-03-14)
Ines Rosignol, Beatriz Villarejo-Zori, Petra Teresak, Elena Sierra-Filardi, Xandra Pereiro, Natalia Rodríguez-Muela, Elena Vecino, Helena L A Vieira, Katharina Bell, Patricia Boya
ABSTRACT

Mitochondrial damage plays a prominent role in glaucoma. The only way cells can degrade whole mitochondria is via autophagy, in a process called mitophagy. Thus, studying mitophagy in the context of glaucoma is essential to understand the disease. Up to date limited tools are available for analyzing mitophagy in vivo. We have taken advantage of the mito-QC reporter, a recently generated mouse model that allows an accurate mitophagy assessment to fill this gap. We used primary RGCs and retinal explants derived from mito-QC mice to quantify mitophagy activation in vitro and ex vivo. We also analyzed mitophagy in retinal ganglion cells (RGCs), in vivo, using different mitophagy inducers, as well as after optic nerve crush (ONC) in mice, a commonly used surgical procedure to model glaucoma. Using mito-QC reporter we quantified mitophagy induced by several known inducers in primary RGCs in vitro, ex vivo and in vivo. We also found that RGCs were rescued from some glaucoma relevant stress factors by incubation with the iron chelator deferiprone (DFP). Thus, the mito-QC reporter-based model is a valuable tool for accurately analyzing mitophagy in the context of glaucoma.

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Sigma-Aldrich
Laminina, 1-2 mg/mL in Tris-buffered saline, 0.2 μm filtered, BioReagent, suitable for cell culture
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Antimycin A from Streptomyces sp.
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Insulina, recombinant, expressed in yeast (proprietary host)
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Oligomycin A, ≥99% (HPLC)
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Deferoxamina, powder, ≥92.5% (TLC)
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Carbonyl cyanide 3-chlorophenylhydrazone, ≥97% (TLC), powder
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HEPES, BioPerformance Certified, suitable for cell culture, ≥99.0%
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Staurosporina, ≥98% (HPLC), film
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Anticorpo anti-Brn-3a, dominio POU della proteina, clone 5A3.2, culture supernatant, clone 5A3.2, Chemicon®
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CORM-A1, ≥95% (NMR)
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1,2-Dilinoleoyl-3-palmitoyl-rac-glycerol, ≥95% (TLC), liquid