This study was supported in part by grants from: the Fight for Sight Leonard &#38; Robert Weintraub Postdoctoral Fellowship (D.Y.S.); BrightFocus Foundation Postdoctoral Fellowship Program in Macular Degeneration Research (M2021010F, D.Y.S.); Department of Defense, Spinal Vision Research Program under Award no. VR180132 (M.S.-G. and L.A.K.); National Eye Institute of the National Institutes of Health under Award no. R01EY027739 (L.A.K.). Acknowledgment is made to the donors of the Macular Degeneration Research M2021010F, a program of the BrightFocus Foundation, for support of this research. Schematics were created with Biorender.com

1. Plating H-RPE in the cell culture plate
<ol>
	<li>Thaw H-RPE in a T25 flask in human RPE medium supplemented with RPE growth medium supplements (4 mM L-glutamine, 25 ng/mL FGF-2, 2% FBS, 30 mg/mL gentamicin, and 15 &#181;g/mL amphotericin).</li>
	<li>Incubate the cells at 37 &#176;C and 5% CO2 in a humidified incubator. Refresh the media the next day and wait for the cells to reach at least 80% confluence before passaging at a ratio of 1:3 into a T75 flask.</li>
	<li>Once confluent, passage the cells using the subculture reagents kit consisting of trypsin/EDTA 0.025% solution, trypsin neutralizing solution, and HEPES buffered saline (pH 7.0-7.6).
	<ol>
		<li>Rinse H-RPE gently with 3 mL of HEPES buffered saline. Then&#160;add 3 mL of trypsin/EDTA and incubate (37 &#176;C and 5% CO2) for 5 min (or until the cells have lifted off the base of the flask, as observed under the microscope). Neutralize the trypsin/EDTA with 3 mL of trypsin neutralizing solution.</li>
		<li>Centrifuge the cells at 200 x g for 3.5 min to form the cell pellet. Carefully aspirate and discard the supernatant.</li>
	</ol>
	</li>
	<li>Resuspend the cells in human RPE medium to a final concentration of 20,000 cells per 100 &#181;L per well.
	<ol>
		<li>Pipette up and down multiple times to ensure that the cell suspension is homogeneous, using a multichannel pipette for ease and consistency of pipetting into the 96-well cell culture microplate. Rest the pipette tip just below the circular rim at the top of the well for an even, homogeneous cell layer. 
		NOTE: No coating is required as the cells adhere well to the microplate.</li>
		<li>Be sure to leave the four corner wells empty of cells (100 &#181;L of media only) to serve as the background correction wells (Figure 1A). 
		NOTE: The microplate is formatted in a typical 96-well plate design; however, the surface area of each well is 0.106 cm2, which is 40% smaller than a standard 96-well plate. At this cell density, the cells should be 100% confluent the next day. It is recommended to perform a cell density optimization experiment first before testing treatments to ensure that basal OCR and ECAR readings are around 50-100 pmol/min and 10-20 mpH/min, respectively.</li>
	</ol>
	</li>
	<li>Leave the cell culture plate at room temperature (RT) for 1 h before placing it back into the incubator (5% CO2, 37 &#176;C, humidified) to help minimize edge effects. Edge effects are changes in media volume in the wells of the peripheral borders of the 96-well plate due to evaporation14. 
	NOTE: The cells will adhere overnight and form a confluent monolayer the next day. H-RPE are matured in the plate for at least 1 month by refreshing half of the growth media every 2-3 days.</li>
	<li>Examine the cells under the microscope before changing the media to check their morphology and pigmentation level. Ensure that the cells are confluent with a characteristic cobblestone-like morphology and acquire pigmentation over time, as seen in the morphology images in Shu et al.7.</li>
</ol>
2. The day before running the assay
<ol>
	<li>Ensure that the sensor cartridge is hydrated the day before the assay by filling each well of the utility plate with 200 &#181;L of deionized water. 
	NOTE: The lid of the utility plate contains fluorescent biosensors for measuring oxygen and pH levels for each well. The sensors are coupled to fiber-optic waveguides that deliver light at various excitation wavelengths and transmit a fluorescent signal through optical filters to photodetectors.</li>
	<li>Place the sensor cartridge submerged in water in the utility plate along with ~20 mL of the calibrant (to warm up for use the next day) in a 37 &#176;C humidified oven (without CO2) overnight. 
	NOTE: The calibrant is a proprietary solution designed for sensor cartridge calibration and is likely similar in composition to phosphate-buffered saline (PBS). The minimum time for cartridge hydration is 4 h, but the best results are obtained with overnight hydration.</li>
	<li>Ensure that the instrument is turned on and start the software to allow the instrument to stabilize to 37 &#176;C overnight (Figure 2). Generally, the instrument can be left on even when not in use.</li>
</ol>
3. Real-time Mito Stress Test using the extracellular flux analyzer
<ol>
	<li>On the day of the assay, replace the water in the utility plate with an equal volume of warmed calibrant at least 45 min prior to running the assay.</li>
	<li>Make the Mito Stress Test assay media using the base medium without phenol red by adding supplements, as shown in Table 1. Warm the media to 37 &#176;C, adjust the pH to 7.4, and vacuum filter the media using a tube top filter unit. For running a full plate, make ~25 mL of assay media.</li>
	<li>Remove the human RPE media and replace it with 180 &#181;L of freshly prepared assay media (step 3.2). Place the cell culture plate in a humidified 37 &#176;C oven (without CO2) for 1 h before starting the assay. 
	NOTE: This is important for de-gassing the cell plate, allowing for CO2 diffusion. Since the cells are no longer in their growth media and no longer in an incubator with CO2, their viability will deteriorate over time, therefore, care should be taken to conduct the assay as efficiently as possible. Care should also be taken to ensure that there are no bubbles in the cell culture plate; if any are present, pop them with a pipette or needle.</li>
	<li>Each sensor cartridge has four reagent delivery ports per well for the injection of test compounds into the wells of the cell culture plate during the assay (Figure 1C,D). Prepare ~3mL of the 10x drug solutions by diluting the drug stocks in the respective assay media according to Table 2. For example, load Port A with 25 &#181;M of oligomycin dissolved in the Mito Stress Test assay media, such that when the drug volume is injected into the well by the instrument, the final concentration that each cell is exposed to is 2.5 &#181;M. Pipette 20 &#181;L of the 10x drug stock into Port A, 22 &#181;L into Port B, and 25 &#181;L into Port C, to achieve the specified final drug concentration in each well. For protocols requiring all four drug ports, pipette 28 &#181;L into Port D. 
	NOTE: Refer to Figure 1B when pipetting the drugs into the drug ports for the proper orientation of Ports A/B/C/D. The notch on the edge of the cartridge should be located in the bottom left corner when loading the drug ports. By pipetting at an angle into each drug port, bubbles can be minimized. If there are any bubbles present, care should be taken to pop them with a pipette or needle.</li>
	<li>Open the Templates tab in the analysis software, select Mito Stress Test, and fill out the Group Definitions.
	<ol>
		<li>Input details on the Injection Strategy (in this case, it is pre-inputted as the Mito Stress Test drugs). Input details regarding the different experimental groups in the assay (e.g., Control or Treatment). Input details on the Assay Media (addition of different supplements and their specific concentrations to the base assay media) and finally add the Cell Type.</li>
		<li>Click on the next tab, Plate Map, where the different groups being examined will be assigned to their specific location on the 96-well plate map. Once this is done, click on the Protocol tab to review the instrument protocol for the default Mito Stress Test protocol. 
		NOTE: The default Mito Stress Test template is ready to use but can be modified in any way to suit the experimental design. For example, the default measuring time is 3 min, but this can be modified to 5 min if desired.</li>
	</ol>
	</li>
	<li>Click on Run the Assay and insert the sensor cartridge that is submerged in the calibrant solution in the utility plate. This process takes around 25 min. In this step, each biosensor is calibrated independently based on the sensor output measured in the calibrant solution of known pH and oxygen concentration.
	<ol>
		<li>Once this is calibrated, remove the utility plate and insert the cell culture plate. 
		NOTE: The cell culture plate is equilibrated first, after which, the instrument begins mixing the assay media and measuring the OCR and ECAR values. This step takes around 1.5 h and is conducted inside the instrument without any intervention from the user. A baseline reading of OCR and ECAR is first established by mixing the assay media for 3 min and then measuring OCR and ECAR for 3 min. The instrument performs three loops of mixing and measuring.</li>
		<li>After the baseline measurement, the instrument automatically injects the Port A drug solution into each well. This is followed by three loops of mixing and measuring (3 min each). The same pattern occurs after each subsequent drug injection (Ports B and C).</li>
	</ol>
	</li>
	<li>Once the run is complete, remove the cell culture plate and sensor cartridge. For quality control purposes, ensure that all the drug ports in the sensor cartridge have been injected by examining the ports to check that no residual drug remains. Discard the sensor cartridge and utility plate as they are single-use items.</li>
	<li>Examine the cells in the cell culture microplate under the microscope to ensure that there is still a confluent monolayer of cells. Discard the assay media and replace it with 60 &#181;L of 1x lysis buffer in each well. 
	NOTE: The lysis buffer is made from 10x lysis buffer diluted to 1x in deionized water with the addition of 1 mM phenylmethylsulfonyl fluoride (PMSF).</li>
	<li>Wrap the edges of the plate in Parafilm to prevent evaporation and place it in a -80 &#176;C freezer to aid in cell lysis overnight, before quantification of the protein content using the BCA assay.</li>
	<li>For data analysis, normalize all data by dividing the OCR and ECAR values by the microgram of protein in each well. Export the Mito Stress Test report generator, which utilizes Excel macros to automatically calculate the Mito Stress Test parameters using the data analysis software. 
	&#8203;NOTE: This can be used to determine basal respiration, maximal respiration, spare respiratory capacity, proton leakage, ATP production, and non-mitochondrial respiration. Definitions of these calculations are listed in Table 3.</li>
</ol>
4. Real-time Glycolytic Stress Test using the extracellular flux analyzer
<ol>
	<li>Perform the Glycolytic Stress Test by following the same steps as the Mito Stress Test, except using the different assay media supplements and drug injections shown in Table 1 and Table 2.</li>
	<li>For data analysis, export the Glycolytic Stress Test report generator, which utilizes Excel macros to automatically calculate the Glycolytic Stress Test parameters from the data analysis software. 
	&#8203;NOTE: This can be used to determine non-glycolytic acidification, glycolysis, glycolytic capacity, and glycolytic reserve. Definitions of these calculations are listed in Table 3.</li>
</ol>
5. BCA protein quantification assay
NOTE: The bicinchonic acid (BCA) protein quantification assay (also known as the Smith assay15) is a copper-based colorimetric assay used to determine the total protein content in a sample. Normalizing OCR and ECAR data to the microgram of protein in each well ensures that different amounts of cells/protein in each well do not skew the readings. The mechanism of the BCA assay is based on two chemical reactions. Firstly, the peptide bonds in proteins reduce cupric ions (Cu2+) to cuprous ions (Cu+), which is a temperature-dependent reaction assisted by&#160;higher temperatures (37 to 60 &#176;C). If there are more peptide bonds, making the amount of Cu2+&#160;proportional to the protein content in the solution16. This reaction results in a&#160;color change from green to an intense purple solution, with a peak absorbance at 562 nm16. The higher the protein content in the sample, the higher the absorbance at this wavelength. The working range of this kit is 20-2,000 &#181;g/mL.
<ol>
	<li>The BCA assay kit contains 1 mL aliquots of bovine serum albumin (BSA) at 2 mg/mL, which serves as the protein concentration reference standard. Prepare a serial dilution in a clear, flat-bottomed 96-well plate by starting from the undiluted 2 mg/mL BSA and subsequently halving the concentration by diluting in deionized water (e.g., 2, 1, 0.5, 0.25, 0.125 mg/mL, etc.). Using a known concentration of protein allows for the calculation of a standard curve, which is used to calculate the protein content of the experimental samples. To improve the accuracy of the calculated protein content measurements, measure absorbance readings of the protein concentration reference standards alongside the experimental samples for each assay.</li>
	<li>Pipette 25 &#181;L of each BSA serial dilution in duplicate into the 96-well plate.</li>
	<li>Pipette 25 &#181;L of the 1x lysis buffer in duplicate into the 96-well plate to serve as the blank.</li>
	<li>Thaw the cell culture plate to room temperature and pipette 25 &#181;L of each cell lysate in duplicate into the 96-well plate.</li>
	<li>Prepare the working reagent in a 50:1 ratio of BCA kit reagents A:B. Mix thoroughly by vortexing to remove any turbidity in the working reagent, so that it is a homogenous green solution. Add 200 &#181;L of the working reagent to each well.</li>
	<li>Protect the plate from light by covering the plate in foil and incubate in a 37 &#176;C oven for 20 min.</li>
	<li>Measure the absorbance at 562 nm in a 96-well plate reader.</li>
	<li>For data analysis, average all the duplicate values. Subtract the blank absorbance levels determined from the 1x lysis buffer wells from the measurements of all the samples. Determine the standard curve by plotting the absorbance of each BSA standard to its known concentration in &#181;g/mL. Use the linear equation derived from the standard curve to determine the protein concentration of the experimental samples.</li>
</ol>

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A., Beaulieu, M. O., Benchorin, G., Vollrath, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+measuring+extracellular+flux+from+intact+polarized+epithelial+monolayers.">Method for measuring extracellular flux from intact polarized epithelial monolayers.</a> Molecular Vision. 24, 425-433 (2018).</li><li>Chichagova, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+iPSC+disease+modelling+reveals+functional+and+structural+defects+in+retinal+pigment+epithelial+cells+harbouring+the+m.3243A+&gt;+G+mitochondrial+DNA+mutation.">Human iPSC disease modelling reveals functional and structural defects in retinal pigment epithelial cells harbouring the m.3243A &gt; G mitochondrial DNA mutation.</a> Scientific Reports. 7 (1), 12320 (2017).</li></ol>

The authors have nothing to disclose.

This optimized protocol for high-resolution respirometry of H-RPE involves the use of BAM15 as the uncoupler instead of the commonly used FCCP. While previous studies on high-resolution respirometry of RPE utilized FCCP9,24, BAM15 appears to induce a more robust induction of maximal respiration levels in H-RPE compared to FCCP. While both FCCP and BAM15 are safe to use in cells, BAM15 is reported to have fewer side effects in normal cells compared to FCCP or carbonylcyanide-3-chlorophenylhydrazone (CCCP)25. Kenwood et al. showed that BAM15 depolarizes mitochondria without impacting plasma membrane potential, thus inducing a sustained maximal mitochondrial respiration rate at low cytotoxicity26. FCCP, on the other hand, depolarizes both mitochondria and the plasma membrane and displays higher cytotoxicity26.
There are several critical steps in the protocol, including ensuring the cells are properly plated in a confluent, even, and homogenous monolayer of RPE in all experimental wells of the microplate. Mature RPE are highly dependent upon OXPHOS for energy generation, and thus the cells should be allowed to mature for at least 1 month to ensure that the RPE generate proper basal and maximal OCR readings during the assay. De-gassing the cell culture plate for 1 h at 37 &#176;C in a&#160;humidified oven (without CO2) before placing it into the instrument is crucial for accurate ECAR readings, as CO2 can impact the pH of the assay media. It is important to remember to hydrate the sensor cartridge the day before the assay to ensure that it provides reliable OCR and ECAR readings on the day of the assay. Care should be taken to properly reconstitute the drugs to be injected and aliquot the reconstituted drug stocks into smaller volumes for long-term storage to minimize freeze/thaw cycles. It is critical to prepare 10x drug solutions that are diluted in the respective assay media (e.g., dilute in Mito Stress Test assay media for the Mito Stress Test) to factor in the dilution from injection of the drug into each well filled with assay media. With each subsequent drug injection there is more media in each well, and thus the volumes of drug being loaded increase with each injection, and care should be taken to follow the volumes specified in the protocol. It is important to perform quality checks on completion of the experiment by examining the sensor cartridge to ensure that no residual drug is evident and observing the cell culture plate under a microscope to ensure that the confluent and homogeneous cell monolayer remains.
Modifications to this protocol include injecting different drugs in the ports and determining how these drugs impact OCR and ECAR readings. One popular modification is to inject an experimental drug of choice as Port A before injecting the usual Mito or Glycolytic Stress Test drugs. This type of protocol provides insights into how an acute injection of the drug of choice impacts the subsequent OXPHOS and glycolytic parameters. Other modifications include examining different cell types; this requires initial troubleshooting of the optimal seeding cell density and optimization of the assay media by ensuring that basal readings range from 50-100 pmol/min for OCR and 10-20 mpH/min for ECAR. The optimal concentrations of the injected drugs need to be determined for each new cell type examined by observing the OCR and ECAR responses to a serial dilution of the drugs.
A key limitation of the protocol is that cell viability diminishes over time, as the cells are not in a CO2 incubator in their normal growth media, and thus the assay should be completed within 3-4 h to ensure maximal cell viability. Furthermore, exposure to mitochondrial toxins injected into each well can further diminish cell viability over time. Once the assay is completed, the cells must be lysed for evaluation of the protein content to normalize OCR and ECAR readings, and thus the same cells cannot be harvested for subsequent molecular biology assays.
Alternatives to the Seahorse for bioenergetic profiling include the Oroboros Oxygraph 2k (O2k)27, BaroFuse28,29, and Resipher (Lucid Scientific)7. The Oroboros O2k is a closed two-chamber respirometer that utilizes S1 Clark-type polarographic oxygen electrodes. While the Oroboros O2k produces highly sensitive measurements of real-time metabolic flux, the device is labor-intensive as the operator is required to manually inject each drug30. The BaroFuse is a novel multichannel microfluidic perfusion system that uses gas pressure to enable multiple parallel perfusion experiments and is linked to an oxygen detection system to measure OCR. The advantage of this flow culture system is that tissue function and viability is maintained, as opposed to the Seahorse where cell viability diminishes over longer assays. The Resipher utilizes highly sensitive optical oxygen sensors to measure OCR whilst the cells are in a 96-well plate in the incubator, hence enabling continuous OCR measurements over several weeks to months. Notably, these instruments do not measure ECAR, and thus the Seahorse has the advantage of simultaneous exploration of both OXPHOS and glycolysis.
Interrogating real-time bioenergetic profiles of OXPHOS and glycolysis is emerging as a key factor in characterizing RPE health and function. High-resolution respirometry enables an efficient means of comparing the metabolic status of normal and diseased RPE, thus opening new avenues of screening drug efficacy for retinal diseases such as AMD and PVR. Future directions for high-resolution respirometry on RPE include optimizing a protocol to examine bioenergetic profiles for highly polarized RPE monolayers grown on transwell filters. Calton et al. (2016) successfully achieved this by cutting a triangular section of the polarized RPE monolayer grown on transwell filters31. Further expansion of the methodology includes examining the bioenergetic profiles of induced pluripotent stem cell-derived RPE (iPSC-RPE), isolated from patients with different retinal degenerative conditions32. Exploration of how pathogenic cytokines involved in AMD and PVR impact the dynamic nature of RPE metabolism can reveal metabolic vulnerabilities that can inform the identification of novel druggable targets.

Retinal pigment epithelial cells (RPE) exist as a monolayer of pigmented epithelial cells strategically located between the photoreceptors and fenestrated endothelium of the choriocapillaris. RPE are highly metabolically active with numerous functions, including (1) phagocytosis of shed photoreceptor discs, (2) recycling of visual pigment to maintain the visual cycle, (3) transport of nutrients, metabolites, ions, and water, (4) absorption of light, (5) protection against photooxidation, (6) secretion of essential factors to support retinal integrity, and (7) formation of the outer blood-retinal barrier1. Degeneration of RPE is associated with metabolic dysfunction and mitochondrial defects, leading to blinding eye diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR)2.
Two key bioenergetic pathways include glycolysis, which occurs in the cytoplasm, and oxidative phosphorylation (OXPHOS), which occurs in mitochondria. During glycolysis, one molecule of glucose is converted to two molecules of pyruvate and a net production of two molecules of adenosine triphosphate (ATP). In contrast to glycolysis, OXPHOS produces far higher levels of ATP (~32-38 molecules of ATP per glucose molecule). Notably, OXPHOS consumes oxygen and requires functional mitochondria to occur, whereas glycolysis occurs in the cytoplasm and does not require oxygen.
Prior to the introduction of fluorescence- or phosphorescence-based techniques to examine mitochondrial respiration, oxygen levels were measured in permeabilized cell suspensions in chambers equipped with a Clark-type oxygen electrode3. While the Clark electrode is much cheaper than fluorescence-based respirometry and works in non-adherent cells, it is relatively low-throughput, with each respiratory run lasting around 15-20 min and requiring far higher quantities of cells for each sample3. Thus, the fluorescence-based respirometry technique has largely replaced the Clark electrode and has become a popular technique in the fields of metabolism and mitochondrial research.
This protocol describes a high-throughput, high-resolution, fluorescence-based respirometry technique that kinetically measures the OXPHOS and glycolytic bioenergetic profiles of living cells. Since the process of OXPHOS consumes oxygen, the bioenergetic profile for OXPHOS is produced by mapping changes in the oxygen consumption rate (OCR) over time4. In this technique, two fluorophores are embedded in the sensor cartridge sleeve which is connected to fiber optic bundles that emit light, exciting the fluorophores. Changes in fluorophore emission are measured by highly sensitive fluorescence sensors and transmitted through the fiber optic bundle to be converted to an OCR readout5. The fluorophore becomes quenched by oxygen, thus enabling the determination of extracellular oxygen levels in the assay media, known as oxygen flux or OCR. The other fluorophore is a pH sensor probe sensitive to changes in proton efflux, which is converted to a measure of extracellular acidification rate (ECAR). During measurements, the fiber optic bundles with embedded fluorophores are lowered to 200 &#181;m above the cell monolayer, creating a transient micro-chamber that enables rapid, real-time readings. Once a 10% change in oxygen or proton levels is detected, the sensors are lifted upward, allowing a larger volume of media to mix with the transient micro-chamber media, restoring OCR and ECAR values back to baseline. Each sensor cartridge is equipped with four ports to allow for the sequential administration of up to four compounds per well during the assay. Measurements can be collected before and after injection of the compounds in each port, revealing key information about the metabolic status of the cells.
Interrogating these two distinct metabolic pathways can yield important discoveries regarding the metabolic status of RPE following exposure to different pathogenic stimuli, and thus can be used to test the efficacy of drugs in restoring the metabolic integrity of RPE6,7,8. The advent of high-throughput respirometry and the availability of specific mitochondrial inhibitors have stimulated more research in defining the bioenergetic profiles of RPE and identifying defects in metabolism and mitochondria during disease states6,7,8,9,10,11,12,13. High-resolution respirometry has highlighted the key role of metabolic reprogramming of RPE in retinal pathologies such as AMD and PVR. Two key cytokines involved in the pathogenesis of AMD and PVR are transforming growth factor-beta 2 (TGF&#946;2) and tumor necrosis factor-alpha (TNF&#945;). The induction of epithelial-mesenchymal transition (EMT) by TGF&#946;2 is accompanied by mitochondrial dysfunction, OXPHOS suppression, and a compensatory increase in glycolytic capacity in RPE6. More recently, the pro-inflammatory cytokine, TNF&#945;, has been shown to induce a significant upregulation of basal OXPHOS and reduced glycolysis in H-RPE7. The administration of dimethyl fumarate significantly suppressed TNF&#945;-induced inflammation in H-RPE and restored mitochondrial morphology and basal bioenergetic profiles7. The divergent metabolic profiles induced by these two growth factors stimulate intriguing mechanistic questions regarding the involvement of metabolic reprogramming in retinal diseases. The following protocol describes the steps for assessing OXPHOS and glycolytic bioenergetic profiles in H-RPE using high-resolution respirometry.

<table><tbody><tr><td>2-Deoxy-D-glucose</td><td>Sigma&amp;nbsp;</td><td>D8375-5G</td><td>Reconstituted in the Glycolytic Assay Media from its powder form and used in the Glycolytic Stress Test (Port C injection)</td></tr><tr><td>96 Well Non-Treated Plate, 5/Pack, Sterile</td><td>VWR</td><td>229597</td><td>BCA Assay 96-well plate</td></tr><tr><td>Antimycin A from&amp;nbsp;Streptomyces&amp;nbsp;sp.</td><td>Sigma</td><td>A8674-25MG</td><td>Inhibitor of Complex III of the electron transport chain (part of the Port C drug injection together with rotenone)</td></tr><tr><td>BAM15</td><td>Sigma</td><td>SML1760-5MG</td><td>Uncoupling agent used for the Mito Stress Test (Port B injection)</td></tr><tr><td>BCA Assay</td><td>ThermoFisher</td><td>23225</td><td>To determine total protein content in each well</td></tr><tr><td>Calibrant Solution</td><td>Seahorse Bioscience&amp;nbsp;</td><td>100840-000</td><td>Solution used to calibrate the probes of the sensor cartridge</td></tr><tr><td>Cell Lysis buffer 10x</td><td>Cell Signaling Technologies</td><td>9803S</td><td>Dilute 10x Cell Lysis Buffer to a 1x solution using deionized water and add 1 mM PMSF before use</td></tr><tr><td>D-(+)-Glucose solution</td><td>Sigma</td><td>G8644-100ML</td><td>Supplement to be added to the Mito Stress Test assay media and also used in the Glycolytic Stress Test (Port A injection)</td></tr><tr><td>DMSO, Cell culture grade</td><td>Sigma-aldrich</td><td>D4540-100ML</td><td>For reconstituting all mitochondrial drugs except for 2DG</td></tr><tr><td>FCCP</td><td>Sigma</td><td>C2920-10MG</td><td>Uncoupling agent used for the Mito Stress Test (Port B injection)</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050061</td><td>Supplement to be added to the assay media for both the Mito and Glycolytic Stress Tests</td></tr><tr><td>HEPES solution</td><td>Sigma</td><td>H0887-100ML</td><td>Supplement to be added to the assay media for both the Mito and Glycolytic Stress Tests</td></tr><tr><td>H-RPE &amp;ndash; Human Retinal Pigment Epithelial Cells</td><td>Lonza</td><td>194987</td><td>Primary human fetal RPE cells</td></tr><tr><td>Oligomycin - CAS 1404-19-9 - Calbiochem</td><td>Sigma</td><td>495455-10MG</td><td>ATP synthase inhibitor used for the Mito Stress Test (Port A injection) and Glycolytic Stress Test (Port B injection)</td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM996</td><td>To seal the plate once the cells are lysed to prevent evaporation in the freezer</td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF) Protease Inhibitor</td><td>Gold Biotechnology Inc</td><td>50-153-2823</td><td>Used at 1 mM for the 1x Cell Lysis Buffer solution</td></tr><tr><td>ReagentPack&amp;nbsp;Subculture Reagents, 100 mL</td><td>Lonza</td><td>CC-5034</td><td>Passaging reagents for primary human fetal RPE cells. Each kit contains 100 mL Trypsin/EDTA, Trypsin Neutralizing Solution, HEPES Buffered Saline</td></tr><tr><td>Rotenone</td><td>Sigma</td><td>R8875-1G</td><td>Inhibitor of Complex I of the electron transport chain (part of the Port C drug injection together with antimycin A)</td></tr><tr><td>RtEGM Retinal Pigment Epithelial Cell Growth Medium BulletKit - RtEBMTM Basal Medium (00195406) and RtEGMTM SingleQuots Supplements (00195407)</td><td>Lonza</td><td>195409</td><td>Media for primary human fetal RPE cells</td></tr><tr><td>Seahorse XFe96 Analyzer</td><td>Agilent</td><td></td><td>High-Resolution Respirometry Instrument</td></tr><tr><td>Seahorse XFe96 FluxPak</td><td>Agilent</td><td>102416-100</td><td>This contains the 96-well Seahorse Cell Culture Microplate, the sensor cartridge and the calibrant solution</td></tr><tr><td>Sodium pyruvate solution</td><td>Sigma</td><td>S8636-100ML</td><td>Supplement to be added to the Mito Stress Test assay media</td></tr><tr><td>Steriflip-GP Sterile Centrifuge Tube Top Filter Unit, 50 mL process volume, 0.22 &amp;micro;m pore size</td><td>Millipore-Sigma</td><td>SCGP00525&amp;nbsp;</td><td>Tube top filter unit for sterile filtration of the assay media</td></tr><tr><td>Synergy H1 Plate Reader</td><td>BioTek</td><td></td><td>Plate Reader for measuring absorbance at 562 nm for the BCA assay</td></tr><tr><td>XF base medium without phenol red&amp;nbsp;</td><td>Agilent</td><td>103335-100</td><td>Base media for running the Seahorse assay</td></tr></tbody></table>

real-time analysis of bioenergetics in primary human retinal pigment epithelial cells using high-resolution respirometry

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Since RPE are highly metabolically-active cells, alterations in their metabolic status reflect changes in their health and function. High-resolution respirometry allows for real-time kinetic analysis of the two major bioenergetic pathways, glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), through quantification of the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), respectively. The following is an optimized protocol for conducting high-resolution respirometry on primary human retinal pigment epithelial cells (H-RPE). This protocol provides a detailed description of the steps involved in producing bioenergetic profiles of RPE to define their basal and maximal OXPHOS and glycolytic capacities. Exposing H-RPE to different drug injections targeting the mitochondrial and glycolytic machinery results in defined bioenergetic profiles, from which key metabolic parameters can be calculated. This protocol highlights the enhanced response of BAM15 as an uncoupling agent compared to carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) to induce the maximal respiration capacity in RPE. This protocol can be utilized to study the bioenergetic status of RPE under different disease conditions and test the efficacy of novel drugs in restoring the basal metabolic status of RPE.

The instrument simultaneously measures both OCR and ECAR for each run. For the Mito Stress Test, parameter calculations are based on OCR readings (Figure 3A), whereas for the Glycolytic Stress Test, parameter calculations are based on ECAR readings (Figure 3B). Figure 3 shows representative graphs for the Mito Stress Test OCR curve over time (Figure 3C) and parameter calculations in the form of bar graphs for H-RPE (Figure 3D). The Glycolytic Stress Test is represented as an ECAR curve over time (Figure 3E), and parameter calculations are displayed in bar graphs for H-RPE (Figure 3F).
Basal respiration provides an understanding of the energetic demands of the cells under basal conditions17. The first drug injection, oligomycin (Oligo), is an ATP synthase inhibitor, and thus, any reduction of OCR following the first drug injection is a measure of ATP-linked respiration18. Any remaining basal respiration following subsequent drug injections is regarded as a proton leak since it is not coupled to ATP synthesis. Increased proton leak may indicate increased mitochondrial uncoupling, which is regulated by uncoupling proteins that are physiologically present but have also been linked to pathologies such as obesity, cancer, type 2 diabetes, and cardiovascular disease19. The second injection is of an uncoupling agent, such as BAM15 or FCCP, to determine the maximal respiration potential of the mitochondria. Uncoupling agents collapse the proton gradient and reduce the proton motive force across the mitochondrial inner membrane. The result is an uninhibited flow of electrons through the electron transport chain (ETC), which elevates the rate of oxygen consumption and causes mitochondrial respiration to reach its maximal capacity20. Spare respiratory capacity (SRC) is the difference between maximal and basal respiration, indicating the capacity of the cells to respond to changes in energetic demands when challenged, indicating cell fitness. Importantly, for the Mito Stress Test in H-RPE, BAM15 is superior to FCCP in enhancing mitochondrial respiration capacity (Figure 4A,B), as maximal respiration and spare respiratory capacity are significantly higher with 10 &#181;M BAM15 compared to 500 nM or 1 &#181;M FCCP. No significant differences were observed between either FCCP dose. The third and final injection of rotenone and antimycin A (Rot/AA) inhibit mitochondrial complexes I and III, respectively, of the ETC, which shuts down mitochondrial respiration; any residual OCR is due to non-mitochondrial sources21.
During glycolysis, one molecule of glucose is converted to two molecules of lactate in the absence of oxygen. The extrusion of lactate from the cell is accompanied by the efflux of one proton per lactate molecule, hence causing acidification of the extracellular space. The flux of proton production in the media is measured by changes in ECAR. Basal glycolysis levels are established first, after which glucose is injected into the assay media, which is devoid of glucose, to induce glycolysis and thus enhance ECAR levels22. Oligomycin is injected to induce the &#34;highest&#34; ECAR as it halts mitochondrial ATP production, thus forcing the cell to derive its ATP through glycolysis. Finally, glycolysis is shut down by adding 2DG, which inhibits hexokinase, the first enzyme of glycolysis23. Any remaining ECAR is likely due to other sources of acidification, such as CO2 production by the TCA cycle during OXPHOS, and is denoted as non-glycolytic ECAR. The glycolytic reserve is calculated as the difference between the &#34;highest&#34; ECAR and the ECAR in the presence of glucose. The glycolytic capacity is the sum of glycolysis and the glycolytic reserve.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64572/64572fig1v2.jpg" /> 
Figure 1: Components of the plate and sensor cartridge. (A) The instrument, data analysis software, and protocol setup interface are shown. In the 96-well culture plate layout, the cells are plated in the blue-colored wells and the four corner wells are marked in black, as they serve as the back correction wells which contain media only (and no cells). (B) There are four drug ports for each well of the sensor cartridge where drugs A, B, C, and D can be loaded. The volumes to pipette into each port are listed based on the calculations for 10x drug stock preparations. (C) The sensor cartridge consists of sensor probes which are placed directly into the calibrant-filled utility plate. <a href="https://www.jove.com/files/ftp_upload/64572/64572fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64572/64572fig2v2.jpg" /> 
Figure 2: Timeline of the assay. The cells are seeded in the cell culture plate. Once ready, the assay involves a 2 day procedure followed by quantification of the protein content using the BCA assay and subsequent data analysis. <a href="https://www.jove.com/files/ftp_upload/64572/64572fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64572/64572fig3v2.jpg" /> 
Figure 3: Representative graphs of the Mito Stress Test and Glycolytic Stress Test. Calculations of (A) Mito Stress Test and (B) Glycolytic Stress Test parameters are depicted in the schematic. (C) Representative oxygen consumption (OCR) curve and (D) Mito Stress Test parameters for H-RPE are shown. (E) Representative extracellular acidification rate (ECAR) curve and (F) Glycolytic Stress Test parameters for H-RPE are shown. Error bars are means &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/64572/64572fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64572/64572fig4v2.jpg" /> 
Figure 4: Comparison of BAM15 versus FCCP as an uncoupling agent. Mito Stress Test on H-RPE comparing the efficacy of inducing maximal respiration using 10 &#181;M BAM15, 500 nM FCCP, or 1 &#181;M FCCP, showing the (A) oxygen consumption rate (OCR) curve and (B) Mito Stress Test parameters. Error bars are means &#177; SEM. **** p &#8804; 0.0001; ns, not significant. <a href="https://www.jove.com/files/ftp_upload/64572/64572fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Test</td>
			<td>Glucose (mM)</td>
			<td>GlutaMax (mM)</td>
			<td>Sodium pyruvate (mM)</td>
			<td>HEPES (mM)</td>
		</tr>
		<tr>
			<td>Mito Stress Test</td>
			<td>25</td>
			<td>2</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Glycolytic Stress Test</td>
			<td>None</td>
			<td>0.5</td>
			<td>None</td>
			<td>1</td>
		</tr>
	</tbody>
</table>
Table 1: Concentration of supplements added to the assay media for the Mito and Glycolytic Stress Tests.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#160;Test</td>
			<td>Port A</td>
			<td>Port B</td>
			<td>Port C</td>
		</tr>
		<tr>
			<td>Mito Stress Test</td>
			<td>Oligomycin 2.5 &#956;M</td>
			<td>FCCP 500 nM OR BAM15 10 &#956;M</td>
			<td>Rotenone 2 &#956;M AND Antimycin A 2 &#956;M</td>
		</tr>
		<tr>
			<td>Glycolytic Stress Test</td>
			<td>Glucose 10 mM</td>
			<td>Oligomycin 2 &#956;M</td>
			<td>2-Deoxyglucose 50 mM</td>
		</tr>
	</tbody>
</table>
Table 2: Concentrations of drug port injections for the Mito and Glycolytic Stress Tests. It is important to note that these are the final concentrations that the cells are exposed to following injection of the drugs into each well. The drugs should be prepared 10x stronger when loading the drug ports.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Term&#160;</td>
			<td colspan="4">Definition&#160;</td>
		</tr>
		<tr>
			<td>Calibrant&#160;</td>
			<td colspan="4">The calibrant is a solution that is used for calibrating the sensor cartridge. Its formulation is proprietary and has a similar composition to PBS.</td>
		</tr>
		<tr>
			<td>Sensor Cartridge&#160;</td>
			<td colspan="4">The sensor cartridge contains two fluorophores embedded in the sensor cartridge sleeve which are connected to fiber optic bundles that emit light, exciting the fluorophores. One fluorophore measures oxygen flux and the other measures proton flux. Each sensor cartridge is also equipped with 4 ports to allow for the sequential administration of up to 4 compounds per well during the assay.</td>
		</tr>
		<tr>
			<td>Utility Plate&#160;</td>
			<td colspan="4">The utility plate (also known as the calibrant plate) is used for calibrating the sensors. The Calibrant solution is placed in the Utility Plate.</td>
		</tr>
		<tr>
			<td>Mito Stress Test</td>
			<td colspan="4">The Mito Stress Test is the name for the assay that provides the mitochondrial respiration bioenergetic profile by plotting changes in OCR over time.</td>
		</tr>
		<tr>
			<td>Glycolytic Stress Test&#160;</td>
			<td colspan="4">The Glycolytic Stress Test is the name of the assay that provides the glycolytic bioenergetic profile by plotting changes in ECAR over time.</td>
		</tr>
		<tr>
			<td>Oxygen Consumption Rate (OCR)</td>
			<td colspan="4">Oxygen consumption rate is a measure of oxygen flux (pmol/min) and indicates the mitochondrial metabolic status.&#160;</td>
		</tr>
		<tr>
			<td>Extracellular Acidification Rate (ECAR)</td>
			<td colspan="4">Extracellular acidification rate is a measure of proton efflux (mpH/min) and indicates the glycolytic metabolic status.</td>
		</tr>
		<tr>
			<td>Wave Software&#160;</td>
			<td colspan="4">The Wave Software is used for programming the assay and subsequent data analysis</td>
		</tr>
		<tr>
			<td>Non-mitochondrial Oxygen Consumption</td>
			<td colspan="4">Minimum rate measurement after Rotenone/antimycin A injection</td>
		</tr>
		<tr>
			<td>Basal Respiration</td>
			<td colspan="4">(Last rate measurement before first injection) &#8211; (Non-Mitochondrial Respiration Rate)</td>
		</tr>
		<tr>
			<td>Maximal Respiration</td>
			<td colspan="4">(Maximum rate measurement after FCCP injection) &#8211; (Non-Mitochondrial Respiration)</td>
		</tr>
		<tr>
			<td>H+ (Proton) Leak</td>
			<td colspan="4">(Minimum rate measurement after Oligomycin injection) &#8211; (Non-Mitochondrial Respiration</td>
		</tr>
		<tr>
			<td>ATP Production</td>
			<td colspan="4">(Last rate measurement before Oligomycin injection) &#8211; (Minimum rate measurement after Oligomycin injection)</td>
		</tr>
		<tr>
			<td>Spare Respiratory Capacity</td>
			<td colspan="4">(Maximal Respiration) &#8211; (Basal Respiration)</td>
		</tr>
		<tr>
			<td>Spare Respiratory Capacity as a %</td>
			<td colspan="4">(Maximal Respiration) / (Basal Respiration) &#215; 100</td>
		</tr>
		<tr>
			<td>Coupling Efficiency</td>
			<td colspan="4">ATP Production Rate) / (Basal Respiration Rate) &#215; 100</td>
		</tr>
		<tr>
			<td>Glycolysis</td>
			<td colspan="4">(Maximum rate measurement before Oligomycin injection) &#8211; (Last rate measurement before Glucose injection)</td>
		</tr>
		<tr>
			<td>Glycolytic Capacity</td>
			<td colspan="4">(Maximum rate measurement after Oligomycin injection) &#8211; (Last rate measurement before Glucose injection)</td>
		</tr>
		<tr>
			<td>Glycolytic Reserve</td>
			<td colspan="4">(Glycolytic Capacity) &#8211; (Glycolysis)</td>
		</tr>
		<tr>
			<td>Glycolytic Reserve as a %</td>
			<td colspan="4">(Glycolytic Capacity Rate) /(Glycolysis) &#215; 100</td>
		</tr>
		<tr>
			<td>Non-glycolytic Acidification</td>
			<td colspan="4">Last rate measurement prior to glucose injection</td>
		</tr>
	</tbody>
</table>
Table 3: List of definitions of key components of the assay.

Watch this Scientific Journal Video about Real-Time Analysis of Bioenergetics in Primary Human Retinal Pigment Epithelial Cells Using High-Resolution Respirometry at JoVE.com

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

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.

Metabolic dysfunction of retinal pigment epithelial cells (RPE) is a key pathogenic driver of retinal diseases such as age-related macular degeneration ...

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Research

JoVE Journal

Biology

2.3K Views.  Schepens Eye Research Institute of Mass. Eye and Ear. Het ondervragen van real-time bio-energetische profielen van oxfos en glycolyse komt naar voren als een belangrijke factor bij het karakteriseren van de gezondheid en functie van RPE. Respirometrie met hoge resolutie maakt een efficiënte manier mogelijk om de metabole status van normale en zieke RPE te vergelijken. Deze techniek heeft het voordeel van gelijktijdige exploratie van zowel oxfos als glycolyse door een meting van de zuurstofverbruikssnelheid, OCR en extracellulaire verzuringssnel...

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

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Pumpless Multi-Channel Flow Culture to Maintain and Assess Tissues and Cells

Maintaining and Assessing Various Tissue and Cell Types of the Eye Using a Novel Pumpless Fluidics System

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell conditions required for the maintenance of function and viability. Toward this end, we have developed a multi-channel flow culture system to maintain tissue and cells in culture and continuously assess function and viability by either in-line sensors and/or collection of outflow fractions. The system combines 8-channel, continuous optical sensing of oxygen consumption rate with a built-in fraction collector to simultaneously measure production rates of metabolites and hormone secretion. Although it is able to maintain and assess a wide range of tissue and cell models, including islets, muscle, and hypothalamus, here we describe its operating principles and the experimental preparations/protocols that we have used to investigate bioenergetic regulation of isolated mouse retina, mouse retinal pigment epithelium (RPE)-choroid-sclera, and cultured human RPE cells. Innovations in the design of the system, such as pumpless fluid flow, have produced a greatly simplified operation of a multi-channel flow system. Videos and images are shown that illustrate how to assemble, prepare the instrument for an experiment, and load the different tissue/cell models into the perifusion chambers. In addition, guidelines for selecting conditions for protocol- and tissue-specific experiments are delineated and discussed, including setting the correct flow rate to tissue ratio to obtain consistent and stable culture conditions and accurate determinations of consumption and production rates. The combination of optimal tissue maintenance and real-time assessment of multiple parameters yields highly informative data sets that will have great utility for research in the physiology of the eye and drug discovery for the treatment of impaired vision.

Author Spotlight: Advancing Eye Physiology Research via a Multi-Channel Flow Culture for Optimal Tissue Maintenance and Real-Time Assessment 

Real-time analysis of live tissue yields important functional and mechanistic data. This paper describes the protocols and critical variables to ensure accurate and reproducible generation of data by a novel and pump-free multi-channel fluidics system that maintains and assesses a wide range of tissue and cell models.

Many in vitro models used to investigate tissue function and cell biology require a flow of media to provide adequate oxygenation and optimal cell ...

Bioengineering

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

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

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

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

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.

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

Neuroscience

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

High-resolution Respirometry to Assess Mitochondrial Function in Permeabilized and Intact Cells

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in biological samples (intact and permeabilized cells, tissues or isolated mitochondria). The high-resolution oxygraph device is equipped with two chambers and uses polarographic oxygen sensors to measure oxygen concentration and calculate oxygen consumption within each chamber. Oxygen consumption rates are calculated using software and expressed as picomoles per second per number of cells. Each high-resolution oxygraph chamber contains a stopper with injection ports, which makes it ideal for substrate-uncoupler-inhibitor titrations or detergent titration protocols for determining effective and optimum concentrations for plasma membrane permeabilization. The technique can be applied to measure respiration in a wide range of cell types and also provides information on mitochondrial quality and integrity, and maximal mitochondrial respiratory electron transport system capacity.

High-resolution respirometry is used to determine mitochondrial oxygen consumption. This is a straightforward technique to determine mitochondrial respiratory chain complexes&#39; (I-IV) respiratory rates, maximal mitochondrial electron transport system capacity, and mitochondrial outer membrane integrity.

A high-resolution oxygraph is a device for measuring cellular oxygen consumption in a closed-chamber system with very high resolution and sensitivity in ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

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.

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 ...

High-Resolution Respirometry to Assess Bioenergetics in Cells and Tissues Using Chamber- and Plate-Based Respirometers

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and assessment of respiratory complexes using diversified substrate-uncoupler-inhibitor titration (SUIT) protocols. Here, the usage of two high-resolution respirometry devices is demonstrated, and a basic collection of protocols applicable for the analysis of cultured cells, skeletal and heart muscle fibers, and soft tissues such as the brain and liver are presented. Protocols for cultured cells and tissues are provided for a chamber-based respirometer and cultured cells for a microplate-based respirometer, both encompassing standard respiration protocols. For comparative purposes, CRISPR-engineered HEK293 cells deficient in mitochondrial translation causing multiple respiratory system deficiency are used with both devices to demonstrate cellular defects in respiration. Both respirometers allow for comprehensive measurement of cellular respiration with their respective technical merits and suitability dependent on the research question and model under study.

Assessing oxidative phosphorylation using high-resolution respirometers has become an integral part of the functional analysis of mitochondria and cellular energy metabolism. Here, we present protocols for the analysis of cellular energy metabolism using chamber and microplate-based high-resolution respirometers and discuss the key benefits of each device.

High-resolution respirometry (HRR) allows monitoring oxidative phosphorylation in real-time for analysis of individual cellular energy states and ...

Novel OCR Monitoring System for Differentiated RPE Cultures

Long-term Monitoring of Oxygen Consumption Rates in Highly Differentiated and Polarized Retinal Pigment Epithelial Cultures

Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for photoreceptor survival. RPE mitochondrial dysfunction is a hallmark of age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world, and proliferative vitreoretinopathy (PVR), a blinding complication of retinal detachments. RPE degenerative conditions have been well-modeled by RPE culture systems that are highly differentiated and polarized to mimic in vivo RPE. However, monitoring oxygen consumption rates (OCR), a proxy for mitochondrial function, has been difficult in such culture systems because the conditions that promote ideal RPE polarization and differentiation do not allow for easy OCR measurements. 
Here, we introduce a novel system, Resipher, to monitor OCR for weeks at a time in well-differentiated RPE cultures while maintaining the RPE on optimal growth substrates and physiologic culture media in a standard cell culture incubator. This system calculates OCR by measuring the oxygen concentration gradient present in the media above cells. We discuss the advantages of this system over other methods for detecting OCR and how to set up the system for measuring OCR in RPE cultures. We cover key tips and tricks for using the system, caution about interpreting the data, and guidelines for troubleshooting unexpected results. 
We also provide an online calculator for extrapolating the level of hypoxia, normoxia, or hyperoxia RPE cultures experience based on the oxygen gradient in the media above cells detected by the system. Finally, we review two applications of the system, measuring the metabolic state of RPE cells in a PVR model and understanding how the RPE metabolically adapts to hypoxia. We anticipate that the use of this system on highly polarized and differentiated RPE cultures will enhance our understanding of RPE mitochondrial metabolism both under physiologic and disease states.

Author Spotlight: Extended Oxygen Consumption Measurement in Retinal Pigment Epithelium Using Resipher

We introduce a novel device for measuring oxygen consumption rates (OCR) in retinal pigment epithelial (RPE) cultures. The device can measure OCR for weeks at a time on RPE grown on standard cell culture plates with standard media while the plates are in a standard cell culture incubator.

Mitochondrial metabolism is critical for the normal function of the retinal pigment epithelium (RPE), a monolayer of cells in the retina important for ...

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

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