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. 2021 May 12;2(2):100533.
doi: 10.1016/j.xpro.2021.100533. eCollection 2021 Jun 18.

Measurement of mitochondrial respiration in the murine retina using a Seahorse extracellular flux analyzer

Trupti Shetty  1   2 , Bomina Park  1   2 , Timothy W Corson  1   2   3
Affiliations

Measurement of mitochondrial respiration in the murine retina using a Seahorse extracellular flux analyzer

Trupti Shetty et al. STAR Protoc. .

Abstract

Mitochondrial metabolism is a critical mechanism that is deregulated in numerous retinal diseases. Here, we elaborate a protocol to quantify oxygen consumption rate as a measure of mitochondrial respiration directly from mouse retinal tissue pieces. Our procedure combines the use of Seahorse extracellular flux technology and ex vivo retinal tissue isolation and is robustly reproducible under different treatment conditions. This protocol allows direct assessment of mitochondrial function in response to drug treatments or genetic manipulation in mouse models. For complete details on the use and execution of this protocol, please refer to Shetty et al. (2020), Sardar Pasha et al. (2021), Kooragayala et al. (2015), and Joyal et al. (2016).

Keywords: Metabolism; Model Organisms; Neuroscience.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Overview of the procedure The key steps are indicated
Figure 2
Figure 2
Retinal dissection and punch preparation Layout of the retinal dissection corresponding to step 2 in the protocol is shown. Incision marks are indicated with a pictorial scissors icon in steps 2b and 2c. After separating the sclera-choroid-RPE layer, the retinal cup is isolated, and four incisions are made. In step 2d, the resulting retinal “flower” is then used to isolate 1 mm sized punches using a biopsy puncher. Regions adjacent to the optic nerve are chosen as displayed.
Figure 3
Figure 3
Output of retinal oxygen consumption rate Plot showing individual oxygen consumption rates (OCR) of five individual retinal punches (taken from both eyes of a normal, untreated animal) plus no tissue control, with indicated arrows showing time points at which FCCP and rotenone/antimycin are injected during the assay
Figure 4
Figure 4
Quantification of mitochondrial respiration parameters Parameters of basal and maximal respiration along with spare respiratory capacity for five individual punches (taken from both eyes of a normal, untreated animal) are graphed using the raw values from the Seahorse instrument (see Figure 1 for a graphical representation of how these parameters are calculated)
See this image and copyright information in PMC

References

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