Name: Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis (Video) | JoVE Journal
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 10 min 19 s
Description: Learn to measure energy metabolism in explanted mouse retinal tissue using extracellular flux analysis. This technique enables real-time assessment of glycolysis and oxidative phosphorylation for retinal disease research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We thank Dr. Alexander Kolesnikov and Dr. Vladimir Kefalov for providing Gnat1-/- mice, for helpful feedback and advice, and for reading the manuscript.
This work was supported by NIH EY025269 (RR), the Diabetes Research Center at Washington University - NIH DK020579 (JRM and RR), a Career Development Award from Research to Prevent Blindness (RR), the Horncrest Foundation (RR), a Career Development Award from JDRF (JRM), NIH DK101392 (CFS), DK020579 (CFS), DK056341 (CFS), and DK114233 (JRM).

Protocols followed the Association for Research in Vision and Ophthalmology Statement for the Use of Animals and were approved by Washington University.
1. Animal Preparation
<ol>
	<li>Keep animals in standard housing with a 12 hours dark to 12 hours light cycle. Begin experiments at standardized times to avoid circadian effects, typically in the morning shortly after lights are turned on.</li>
</ol>
2. Solution Preparation
<ol>
	<li>Prepare base media by dissolving 8.4 mg of powdered media in double-distilled H2O (ddH2O) and adjust pH to 7.4 with either HCl or NaOH, to a final volume of 1 L. Filter sterilize this solution with a 0.22 &#181;m tissue culture filter.
	<ol>
		<li>Add 1 M glucose and 100 mM sodium pyruvate to the media to achieve final concentrations of 5 mM glucose and 1 mM pyruvate.</li>
		<li>Place a 50 mL aliquot of the base media in a 37 &#176;C water bath.</li>
	</ol>
	</li>
	<li>Prepare lysis solution, for tissue quantitation at the end of the flux analysis, by adding tris base to 10 mM, polyethylene glycol tert-octylphenyl ether to 0.2% (v/v), and EDTA to 1 mM, all in ddH2O. Mix until all components completely dissolved and adjust pH to 8.0.</li>
	<li>For the mitochondrial stress protocol, prepare an 11 &#181;M stock of oligomycin, an ATP Synthase inhibitor, dissolved in base media to target a final concentration of 1 &#181;M in the well. Prepare an 11 &#181;M stock of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), an uncoupling agent, dissolved in base media to target a final concentration of 1 &#181;M in the well. Prepare a cocktail of the electron transport chain inhibitors, rotenone and antimycin A (RAA) by adding rotenone to 11 &#181;M and antimycin A to 22 &#181;M, dissolved in base media to target a final concentration of 1 &#181;M rotenone and 2 &#181;M antimycin A in the well.</li>
	<li>For the glycolysis protocol, prepare a 220 mM stock of glucose dissolved in base media, to target a final concentration of 20 mM in the well. Also prepare a 1.1 M stock of 2-deoxyglucose (2-DG), a competitive inhibitor of glucose and glycolytic antagonist, by dissolving it in base media. This will target a final concentration of 100 mM 2-DG in the well.</li>
</ol>
3. Instrument Calibration
<ol>
	<li>To calibrate the instrumentation for an extracellular flux assay, add 1 mL of calibration solution to each well of the sensor cartridge (24 wells total) and incubate at 37 &#176;C in a CO2-free incubator overnight (or at least 8 h).</li>
	<li>Load additives for the mitochondrial stress protocol or the glycolysis protocol into each injector port, A through D, on the sensor cartridge, adjusting for volume changes in the well. 
	Note: As an example, a typical assay would include 45 &#181;L of an additive into the first injector port, 49.5 &#181;L into the second, 54.5 &#181;L into the third, and 60 &#181;L into the last, assuming an initial well volume of 450 &#181;L.</li>
	<li>During the first several experiments, load base media into port A to gauge how much tissue movement/artefact occurs after a port injection.</li>
	<li>Allow the loaded sensor plate to incubate at 37 &#176;C in a non-CO2 incubator for at least 60 min.</li>
</ol>
4. Isolation of Fresh Mouse Retinal Tissue
Note: This step is adapted from the technique of Dr. Barry Winkler15.
<ol>
	<li>After administering deep anesthesia using a standard ketamine/xylazine cocktail, euthanize mice by cervical dislocation.</li>
	<li>Use a pair of medium-sized curved forceps to grasp the posterior globe at the optic nerve and gently apply forward pressure to proptose the eye (Figure 1A).
	<ol>
		<li>With a clean razor blade, create a limbus to limbus incision across the cornea using a single, deliberate pass of the blade.</li>
		<li>Using fine, angled McPherson-style forceps, pinch the posterior globe to express the lens and anterior hyaloid membranes out of the corneal incision. Discard these tissues.</li>
		<li>Repeat the pinching maneuver with forceps to express the neural retina.</li>
		<li>Using the forceps like a spoon to lift the retina, rather than to grasp the tissue, transfer the retina away from the corneal incision and place directly into warm media in a 3-cm dish or a 6-well tissue culture cluster plate.</li>
	</ol>
	</li>
	<li>Use two pairs of fine, angled McPherson-style forceps to gently dissect remaining vitreous from the retina. This is best done by grasping the vitreous at the periphery of the retinal cup and pulling towards the center, with a final disinsertion of the vitreous body from the center as a whole. Using the forceps, remove any residual retinal pigment epithelium from the photoreceptor surface of the retina.
	<ol>
		<li>Cut a P1000 pipet tip with a razor blade to create a ~4 mm opening to prevent undue trauma to the delicate retinal tissue during transfer maneuvers.</li>
		<li>Transfer the isolated retinal tissue into fresh media using a cut P1000 pipet tip.</li>
		<li>Cut 1 mm punches of retina around the optic nerve with a 1 mm biopsy punch equipped with a plunger to dislodge the tissue in case it becomes lodged into the bore (Figure 1B). Set the retinal punches aside in clean media kept on a 37 &#176;C block or heating pad.</li>
	</ol>
	</li>
</ol>
5. Assay Protocol
<ol>
	<li>Transfer individual punches to a 24-well islet capture microplate using a cut P1000 tip, aspirating 450 &#181;L of media along with the tissue.</li>
	<li>Use fine, straight forceps to gently manipulate punches into the center of the microplate. Keep punches orientated in the same direction (e.g., ganglion cell side upwards).
	<ol>
		<li>Rest the capture plate on a heating pad or heating block set to 37 &#176;C as these steps are repeated for the remaining samples. 
		Note: For each experiment, set aside 3-4 blank wells with 450 &#181;L of base media to serve as negative controls. In the remaining 20-21 wells, test each biologic replicate in triplicate. Therefore, each 24-well plate will allow for recording from 6-7 different animals or treatment conditions.</li>
	</ol>
	</li>
	<li>Avoiding air bubbles, gently position the islet capture mesh inserts into each well using forceps and secure the inserts with a metal plunger, or with a cut P1000 pipet tip (Figure 1C). 
	Note: Avoid excessive tissue movement during this step to maintain retinal punch position within the center of the sample microchamber. Air bubbles severely distort OCR readings. Although the microchamber has adequate depth to accommodate typical mouse retina (Figure 1C), occasionally machining-defects in the mesh inserts may cause tissues to become overly compressed during screen insertion. If this happens, simply make a note of the crushed tissue and exclude that sample from the final analysis.</li>
	<li>Incubate the tissue plate in a 37 &#176;C CO2-free incubator for at least 60 min.</li>
	<li>Program the extracellular flux analyzer using Mix, Wait, Measure, and Repeat commands. As an example, a typical experiment with retinal tissue may include the following: Mix 2 minutes, Wait 2 minutes, and Measure 5 minutes. Repeat the experiment with these three steps between 5-8 times (cycles) for a baseline recording, and after injection of each compound being tested during the run.</li>
	<li>Press the program START button on the extracellular flux analyzer and follow the instructions on the screen to insert the sensor cartridge for calibration.</li>
	<li>At the end of the calibration, follow instructions on the screen to replace the calibrant plate with the plate containing the retinal samples.</li>
	<li>Allow the program to run, as programmed. At the completion of the run, follow instructions on the screen to eject the tissue plate. View the results of the run and store the data file.</li>
	<li>With a bent 20 gauge needle and forceps, remove all mesh inserts from the wells, leaving the retinal punch behind.</li>
	<li>Carefully aspirate the media from the tissue and wash the tissue twice with 0.5 mL of cold phosphate-buffered saline (PBS). Aspirate the PBS after washing.</li>
	<li>Add 100 &#181;L of lysis buffer to each well and pipet up and down to homogenize tissue.</li>
	<li>Quantitate input levels based on total double-stranded DNA (dsDNA) or total protein content. 
	Note: The following is based on a commercially available dsDNA assay.</li>
	<li>Dilute lysed samples 1:1 by adding 100 &#181;L of Tris-EDTA (TE) buffer and transfer 100 &#181;L of the diluted sample to a 96 well plate.
	<ol>
		<li>Add 100 &#181;L of detection buffer into each well. After mixing for 2-5 minutes, quantitate fluorescence after excitation at 480 nm and emission at 520 nm, comparing to a standard curve.</li>
		<li>Normalize the raw extracellular flux tracing to DNA content within the well. 
		Note: In the results presented here, samples are normalized to 50 ng dsDNA - a typical amount in a 1 mm retinal punch. Therefore, absolute values presented here may be compared to studies presenting data &#34;per retinal punch&#34;. Normalize tracings to an internal standard, such as the baseline run at a glucose concentration of 5 mM.</li>
	</ol>
	</li>
</ol>

<ol><li>Wong-Riley, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Energy+metabolism+of+the+visual+system%5BTitle%5D)+AND+%22Eye+and+brain%22%5BJournal%5D)">Energy metabolism of the visual system.</a> Eye and brain. 2, 99-116 (2010).</li>
<li>Ames, A. 3rd, Li, Y. Y., Heher, E. C., Kimble, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Energy+metabolism+of+rabbit+retina+as+related+to+function%3A+high+cost+of+Na%2B+transport%5BTitle%5D)+AND+%22The+Journal+of+neuroscience%3A+the+official+journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">Energy metabolism of rabbit retina as related to function: high cost of Na+ transport.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 12 (3), 840-853 (1992).</li>
<li>Warburg, O. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/13298683">On the origin of cancer cells.</a> Science. 123 (3191), 309-314 (1956).</li>
<li>Wubben, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photoreceptor+metabolic+reprogramming+provides+survival+advantage+in+acute+stress+while+causing+chronic+degeneration%5BTitle%5D)+AND+%22Scientific+reports%22%5BJournal%5D)">Photoreceptor metabolic reprogramming provides survival advantage in acute stress while causing chronic degeneration.</a> Scientific reports. 7 (1), 17863(2017).</li>
<li>Du, J., Linton, J. D., Hurley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Probing+Metabolism+in+the+Intact+Retina+Using+Stable+Isotope+Tracers%5BTitle%5D)+AND+%22Methods+in+enzymology%22%5BJournal%5D)">Probing Metabolism in the Intact Retina Using Stable Isotope Tracers.</a> Methods in enzymology. 561, 149-170 (2015).</li>
<li>Felder, A. E., Wanek, J., Tan, M. R., Blair, N. P., Shahidi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Method+for+Combined+Retinal+Vascular+and+Tissue+Oxygen+Tension+Imaging%5BTitle%5D)+AND+%22Scientific+reports%22%5BJournal%5D)">A Method for Combined Retinal Vascular and Tissue Oxygen Tension Imaging.</a> Scientific reports. 7 (1), 10622(2017).</li>
<li>Hurley, J. B., Lindsay, K. J., Du, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glucose%2C+lactate%2C+and+shuttling+of+metabolites+in+vertebrate+retinas%5BTitle%5D)+AND+%22Journal+of+neuroscience+research%22%5BJournal%5D)">Glucose, lactate, and shuttling of metabolites in vertebrate retinas.</a> Journal of neuroscience research. 93 (7), 1079-1092 (2015).</li>
<li>Winkler, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycolytic+and+oxidative+metabolism+in+relation+to+retinal+function%5BTitle%5D)+AND+%22The+Journal+of+general+physiology%22%5BJournal%5D)">Glycolytic and oxidative metabolism in relation to retinal function.</a> The Journal of general physiology. 77 (6), 667-692 (1981).</li>
<li>Pelletier, M., Billingham, L. K., Ramaswamy, M., Siegel, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+flux+analysis+to+monitor+glycolytic+rates+and+mitochondrial+oxygen+consumption%5BTitle%5D)+AND+%22Methods+in+enzymology%22%5BJournal%5D)">Extracellular flux analysis to monitor glycolytic rates and mitochondrial oxygen consumption.</a> Methods in enzymology. 542, 125-149 (2014).</li>
<li>Joyal, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Retinal+lipid+and+glucose+metabolism+dictates+angiogenesis+through+the+lipid+sensor+Ffar1%5BTitle%5D)+AND+%22Nature+medicine%22%5BJournal%5D)">Retinal lipid and glucose metabolism dictates angiogenesis through the lipid sensor Ffar1.</a> Nature medicine. 22 (4), 439-445 (2016).</li>
<li>Kooragayala, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+Oxygen+Consumption+in+Retina+Ex+Vivo+Demonstrates+Limited+Reserve+Capacity+of+Photoreceptor+Mitochondria%5BTitle%5D)+AND+%22Investigative+ophthalmology+%26+visual+science%22%5BJournal%5D)">Quantification of Oxygen Consumption in Retina Ex Vivo Demonstrates Limited Reserve Capacity of Photoreceptor Mitochondria.</a> Investigative ophthalmology & visual science. 56 (13), 8428-8436 (2015).</li>
<li>Pearsall, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((PPARalpha+is+essential+for+retinal+lipid+metabolism+and+neuronal+survival%5BTitle%5D)+AND+%22BMC+biology%22%5BJournal%5D)">PPARalpha is essential for retinal lipid metabolism and neuronal survival.</a> BMC biology. 15 (1), 113(2017).</li>
<li>Nicholls, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bioenergetic+profile+experiment+using+C2C12+myoblast+cells%5BTitle%5D)+AND+%22Journal+of+visualized+experiments%22%5BJournal%5D)">Bioenergetic profile experiment using C2C12 myoblast cells.</a> Journal of visualized experiments. (46), (2010).</li>
<li>Lobanova, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transducin+translocation+in+rods+is+triggered+by+saturation+of+the+GTPase-activating+complex%5BTitle%5D)+AND+%22The+Journal+of+neuroscience%3A+the+official+journal+of+the+Society+for+Neuroscience%22%5BJournal%5D)">Transducin translocation in rods is triggered by saturation of the GTPase-activating complex.</a> The Journal of neuroscience: the official journal of the Society for Neuroscience. 27 (5), 1151-1160 (2007).</li>
<li>Winkler, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+electroretinogram+of+the+isolated+rat+retina%5BTitle%5D)+AND+%22Vision+research%22%5BJournal%5D)">The electroretinogram of the isolated rat retina.</a> Vision research. 12 (6), 1183-1198 (1972).</li>
<li>Jastroch, M., Divakaruni, A. S., Mookerjee, S., Treberg, J. R., Brand, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+proton+and+electron+leaks%5BTitle%5D)+AND+%22Essays+in+biochemistry%22%5BJournal%5D)">Mitochondrial proton and electron leaks.</a> Essays in biochemistry. 47, 53-67 (2010).</li>
<li>Divakaruni, A. S., Paradyse, A., Ferrick, D. A., Murphy, A. N., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+and+interpretation+of+microplate-based+oxygen+consumption+and+pH+data%5BTitle%5D)+AND+%22Methods+in+enzymology%22%5BJournal%5D)">Analysis and interpretation of microplate-based oxygen consumption and pH data.</a> Methods in enzymology. 547, 309-354 (2014).</li>
<li>Du, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phototransduction+Influences+Metabolic+Flux+and+Nucleotide+Metabolism+in+Mouse+Retina%5BTitle%5D)+AND+%22The+Journal+of+biological+chemistry%22%5BJournal%5D)">Phototransduction Influences Metabolic Flux and Nucleotide Metabolism in Mouse Retina.</a> The Journal of biological chemistry. 291 (9), 4698-4710 (2016).</li>
</ol>

The authors have nothing to disclose.

OCR and ECAR are readily measured in explanted retinal tissue using a bioanalyzer using the described techniques. This method departs from those of other groups in several critical steps. Retinal tissues are isolated through a large corneal incision without enucleating the globe, as originally described by Winkler15. This method of retinal isolation allows for a rapid transfer from the living eye into the tissue capture plate (often within 5 minutes). Tissues are kept at 37 &#176;C throughout the ...

The retina is among the most energy-demanding tissues in the central nervous system1. Like most tissues, it generates adenosine triphosphate (ATP) via glycolysis in the cytosol or via oxidative phosphorylation in mitochondria. The energetic advantage of oxidative phosphorylation over glycolysis to produce ATP from one molecule of glucose is clear: 36 molecules of ATP generated from the former vs. 2 molecules of ATP generated from the latter. Accordingly, retinal neurons primarily depend on mitochondrial respiration for energy supply and this is reflected by their high density of mitochondria2. Yet, the retina also relies heavily on glycolytic machinery even when oxygen is abundant. This process of aerobic glycolysis was originally described in cancer cells by Otto Warburg3, who once noted that the retina was the only post-mitotic tissue capable of this form of metabolism4. Since those initial observations, many post-mitotic tissues have been described to engage in varying degrees of glycolysis in addition to oxidative phosphorylation to meet their ATP demands.
Phototransduction, visual pigment recycling, biosynthesis of photoreceptor outer segments, and synaptic activity are all energy demanding processes in photoreceptors, the predominant neuronal subclass in the retina. But the need to actively transport ions against their electrical and concentration gradients is by far the most energetically consuming process in neurons1. Photoreceptors are peculiar neurons in the sense that they are depolarized in the absence of stimulation (i.e., in the dark), whereas a light stimulus triggers channel closure and subsequent hyperpolarization. Therefore, in the dark, the retina consumes large quantities of ATP to maintain its depolarization or &#34;dark current&#34; as it is commonly called. From an adaptive standpoint, a major challenge in supplying these vast quantities of ATP is the need for organisms to maintain visual clarity through the optical axis. The inverted retinal architecture seen in modern creatures is the dominant solution, as it keeps the dense capillary network supplying photoreceptors away from the path of light. But this marvel of natural bioengineering places the retina at a precipice in terms of metabolic reserve. Even small insults to retina can potentially disrupt the delicate balance of energy supply to demand, and visual dysfunction or frank blindness may ensue quickly.
Given the unique energetic demands of the neural retina, coupled with its tight restriction of vascular supply, accurate measurement of ATP consumption in the retina and its changes during disease could have profound implications in understanding and treating blinding conditions such as retinitis pigmentosa and diabetic retinopathy. Traditionally, these measurements require costly, custom-designed equipment with most studies emerging from a handful of laboratories entirely dedicated to measurements of metabolic activity2,5,6,7,8. Techniques include individual assays for specific metabolites, tracer studies using radio-labeled precursors, oxygen consumption recording using Clark electrodes, and metabolomic profiling9.
With advances in high-throughput technology and increased availability of commercial devices, techniques to record retinal metabolism are increasingly accessible and affordable. The method described here measures both oxidative phosphorylation and glycolysis in retina using explanted tissue and a commercially-available extracellular flux analyzer9,10,11,12. This analyzer separately records oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR), serving as indirect indicators of oxidative phosphorylation and glycolysis, respectively13. These measurements are done by a probe submersed within a microchamber created over the tissue of interest. This adaptation of previously published methods uses a capture plate originally designed for pancreatic Islets of Langerhans to record metabolic activity in small, circular sections of mouse retina. Multiple pharmacologic exposures can be delivered to the tissue during the course of a single recording because the system contains 4 injection ports for each sample well. Using this system with separate protocols optimized for ECAR and OCR recordings, the responses of wild-type retinas can be compared to retinas lacking transducin (Gnat1-/-), a cause of congenital stationary night blindness in humans14.

<table><tbody><tr><td>Seahorse XF24 Extracellular Flux Analyzer</td><td>Agilent, Santa Clara, CA</td><td></td><td></td></tr><tr><td>Seahorse XF24 Islet Capture FluxPak (includes: Islet Capture Microplate, Sensor Cartridge and Calibrant Solution)</td><td>Agilent, Santa Clara, CA</td><td>101174-100</td><td>Includes islet capture microplate, sensor cartridge and calibrant solution</td></tr><tr><td>RPMI 1640 Media (Powdered medium)</td><td>Millipore-Sigma</td><td>R1383</td><td>RPMI 1640 Media with L-Glutamine and without glucose or sodium bicarbonate</td></tr><tr><td>D-Glucose</td><td>Millipore-Sigma</td><td>G8270</td><td>1M D-Glucose filtered, for media preparation</td></tr><tr><td>Sodium pyruvate</td><td>Corning</td><td>25000CI</td><td>100 mM sodium pyruvate</td></tr><tr><td>Antimycin-A</td><td>Millipore-Sigma</td><td>A8674</td><td>Mitochondrial stress protocol component</td></tr><tr><td>FCCP</td><td>Millipore-Sigma</td><td>C2920</td><td>Mitochondrial stress protocol component</td></tr><tr><td>Rotenone</td><td>Millipore-Sigma</td><td>R8875</td><td>Mitochondrial stress protocol component</td></tr><tr><td>2-deoxyglucose</td><td>Millipore-Sigma</td><td>D6134</td><td>Glycolysis protocol component</td></tr><tr><td>1 mm skin biopsy punches with plunger</td><td>Integra-Miltex</td><td>33-31AA-P/25</td><td>Explanting retinal tissue tool</td></tr><tr><td>Dumont Mini-Forceps Straight</td><td>Fine Science Tools</td><td>11200-10</td><td>Explanting retinal tissue tool</td></tr><tr><td>Dumont Medical #5/45 Forceps- Angled 45 degrees</td><td>Fine Science Tools</td><td>11253-25</td><td>Explanting retinal tissue tool</td></tr><tr><td>Dumont #7 Forceps - Curved</td><td>Fine Science Tools</td><td>11271-30</td><td>Explanting retinal tissue tool</td></tr><tr><td>Quant-iT Picogreen dsDNA Assay Kit</td><td>Fisher Scientific</td><td>P7589</td><td>Loading normalization assay</td></tr><tr><td>Trizma base (Tris base)</td><td>Millipore-Sigma</td><td>T6066</td><td>Component of lysis buffer</td></tr><tr><td>Triton X-100 (polyethylene glycol tert-octylphenyl ether)</td><td>Millipore-Sigma</td><td>X100</td><td>Component of lysis buffer</td></tr><tr><td>0.5M EDTA pH 8.0</td><td>Ambion</td><td>AM9262</td><td>Component of lysis buffer</td></tr><tr><td>C57BL/6J mice&amp;nbsp;</td><td>Jackson Laboratories</td><td>&amp;nbsp;Strain 000664</td><td>Animals</td></tr><tr><td>Gnat1&lt;sup&gt;-/-&lt;/sup&gt; and background-matched Gnat1&lt;sup&gt;+/+&lt;/sup&gt;&amp;nbsp;</td><td>Vladimir Kefalov, PhD; Washington University School of Medicine</td><td>Animals</td><td></td></tr></tbody></table>

measurement of energy metabolism in explanted retinal tissue using extracellular flux analysis

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

Using the described techniques (summarized in Figure 1), retinal explants from 8 week-old wild type (WT) mice were compared to age- and background-matched transducin null mice (Gnat1-/-). Because Gnat1-/- animals lack the machinery to close cyclic-nucleotide gated ion channels in response to light stimuli, their rod photoreceptors remain depolarized even in light14. The subsequent need t...

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

measurement-energy-metabolism-explanted-retinal-tissue-using

Research

JoVE Journal

Biology

This protocol provides a biochemical approach for measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of primary leukemic cells with extracellular flux assay. The assessment of the metabolic profile of these cells helps to characterize their demands, leading to personalized therapy.

Extracellular Flux Assay: A Method for Measuring Metabolic Profile of Cells

This video demonstrates the method to analyze energy metabolism in activated human NK cells using an extracellular flux analyzer. Different inhibitors of the electron transport chain complexes impact ATP synthesis, which is measured through the oxygen consumption rate or OCR, providing insights into cellular metabolism and bioenergetics.

Real-Time Monitoring of Energy Metabolism in Human NK Cells Using an Extracellular Flux Analyzer

Metabolic reprogramming is a characteristic and prerequisite for M1 and M2 macrophage polarization. This manuscript describes an assay for the measurement of fundamental parameters of glycolysis and mitochondrial function in mouse bone marrow-derived macrophages. This tool can be applied to investigate how particular factors affect the macrophage’s metabolism and phenotype.

Metabolic Characterization of Polarized M1 and M2 Bone Marrow-derived Macrophages Using Real-time Extracellular Flux Analysis

This protocol provides a cost-effective approach to isolate and characterize mouse primary renal tubular cells that can subsequently be sub-cultured to assess renal biological functions ex vivo, including mitochondrial bioenergetics.

Isolation, Characterization, And High Throughput Extracellular Flux Analysis of Mouse Primary Renal Tubular Epithelial Cells

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

The metabolic status of human retinal pigment epithelial cells (H-RPE) reflects their health and function. Presented here is an optimized protocol for examining the real-time metabolic flux of H-RPE using high-resolution respirometry.

Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry

Here, we describe in detail methods for extracting macrophages from the bone marrow, spleen, and infarcted heart, and subsequently assessing metabolic flux in live cells.

Analyzing Ex Vivo Metabolic Flux in Splenic and Cardiac Macrophages and Bone Marrow Monocytes

This protocol describes a cost-effective way to perform a glycolysis stress test to quantify metabolic reprogramming in primary mouse microglia. After isolation and inflammatory stimulation, extracellular acidification rate (ECAR) is measured using freshly prepared, pH-adjusted reagents, followed by protein normalization to enable accurate and reproducible assessment of glycolytic shifts.

Investigating Glycolysis in Primary Microglia Using Extracellular Flux Assay

In this article, we describe a detailed protocol for quantifying the oxygen consumption rate and extracellular acidification rate of ex vivo adult zebrafish ventricles to characterize their oxidative and glycolytic metabolic capacity.

Assessing the Metabolic Activity of Whole Regenerating Zebrafish Ventricles Ex Vivo Using an Extracellular Flux Assay

Learn to measure energy metabolism in explanted mouse retinal tissue using extracellular flux analysis. This technique enables real-time assessment of glycolysis and oxidative phosphorylation for retinal disease research.

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

Explore More Videos