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

Summary

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.

Abstract

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.

Introduction

The retinal pigment epithelium (RPE) is a monolayer of functionally postmitotic, highly polarized epithelial cells that form a barrier between light-sensitive photoreceptors in the retina and their blood circulation, a capillary bed termed the choriocapillaris. Like the role of glia-supporting neurons, the RPE carries out myriad functions to support photoreceptors, including phagocytosis of photoreceptor outer segments, transport of nutrients and metabolic support for photoreceptors, and secretion of essential growth factors, all critical for maintaining visual function.

Degeneration of the RPE underlies several common degenerative disorders of vision. In age-related macular degeneration (AMD), one of the most common causes of incurable vision loss in the world, the RPE dies, and overlying photoreceptors therefore suffer a secondary degeneration. In proliferative vitreoretinopathy (PVR), the RPE instead exits its normally quiescent postmitotic state, proliferating and dedifferentiating into a mesenchymal state (a so-called epithelial-to-mesenchymal transition [EMT]) with alterations in its metabolism. RPE dedifferentiation causes a loss of RPE support to photoreceptors while also triggering a more fibrotic state. This results in both photoreceptor degeneration and RPE-induced scarring, both of which trigger vision loss^1,2.

A major part of the RPE's support to photoreceptors is metabolic, and metabolic dysregulation is a critical factor in numerous retinal diseases, including AMD and PVR. The RPE serves as a regulatory barrier between photoreceptors and their source of oxygen and nutrients, the choriocapillaris. Thus, what the RPE chooses to metabolize versus what the RPE chooses to pass through from the choriocapillaris to the photoreceptors strongly governs photoreceptor metabolism and survival. Numerous studies have shown that the RPE is heavily dependent on mitochondrial metabolism for its normal health, and that photoreceptors instead rely heavily on glycolysis³. This has introduced the concept of complementary, intertwined metabolic states between photoreceptors and the RPE. Specifically, the RPE reduces its metabolism of preferred photoreceptor metabolic substrates and instead utilizes the byproducts of photoreceptor metabolism combined with the metabolites that photoreceptors do not consume. In diseases such as PVR and AMD, evidence strongly suggests that the RPE becomes more glycolytic and less dependent on mitochondrial metabolism; this shift towards RPE glycolysis may starve photoreceptors of metabolites it needs, triggering degeneration^4,5,6. Given how interdependent RPE and photoreceptor metabolism are and how much altered metabolism underlies retinal disease, there is strong interest in modeling and manipulating RPE metabolism for therapeutic purposes.

While studying RPE mitochondrial metabolism in vivo is ideal, many aspects of RPE mitochondrial metabolism can only be practically probed in an in vitro culture system. Significant progress towards high-fidelity RPE cultures has been made in the past several decades, to the point that the most carefully groomed RPE cultures are now being used for cell replacement therapy in human clinical trials⁷. To maintain such high-fidelity cultures, the RPE needs to be grown on particular substrates in particular media for months prior to experimentation. With these conditions, RPE cultures are maximally differentiated and polarized, approximating in vivo RPE. Unfortunately, there is no equipment currently available that can measure mitochondrial metabolism specifically from the RPE in vivo. While oxygen monitoring of the retinal capillary network has been achieved in vivo using electron paramagnetic resonance (EPR) oximetry⁸, this is not possible for RPE analysis. Differences between RPE metabolism in vivo and in vitro are not well-described, but RPE cultures have been shown to have high mitochondrial activity, similar to RPE in vivo^3,9, suggesting significant insight into RPE mitochondrial metabolism can be gained using high-fidelity RPE cultures.

As all mitochondrial metabolism leads to oxygen consumption, measuring RPE oxygen consumption rates (OCR) is a faithful proxy for mitochondrial metabolism. Measuring OCR in RPE cultures has been difficult, as the conditions that promote maximal RPE polarization and differentiation often preclude long-term accurate OCR measurements with currently available techniques, such as the Seahorse Analyzer. In this methods paper, a novel device, termed the Resipher (hereafter referred to as "the system"), is introduced, which allows continuous measurement of OCR over weeks in RPE grown under conditions that maximally promote polarization and differentiation. The ease with which OCR can be measured by this system in RPE culture conditions that maximally promote RPE differentiation and polarization is unique among existing OCR-measuring devices.

This paper provides tips and tricks for using the system with RPE cultures, followed by a demonstration of two particular applications. First, RPE EMT, mimicking PVR, is triggered by exposure to transforming growth factor-beta (TGFβ)^1,10,11,12. The system is used to monitor how RPE metabolism evolves during the EMT process. Second, the role of hypoxia in RPE metabolism is explored using this system. Hypoxia is an important contributor to the pathogenesis of AMD, as the choriocapillaris thins with age^13,14. Combining this system with hypoxia chambers allows one to model altered RPE mitochondrial metabolism with the subtle hypoxia that accompanies aging. Finally, an online calculator using Resipher data is introduced to allow one to determine whether RPE cultures are in hypoxic, normoxic, or hyperoxic conditions. Such information is critical for drawing any conclusions about RPE metabolism from in vitro RPE culture studies.

Protocol

For protocols to establish human primary or iPSC-RPE cultures, see the following references^15,16,17,18. The acquisition and use of human tissue for these protocols were reviewed and permitted by the University of Michigan Institutional Review Board (HUM00105486).

1. General application of the system to RPE culture

1. Plate human induced pluripotent stem cell (iPSC) derived-RPE or primary human RPE cells in system-compatible 96-well plates.
   1. Assuming already-existing mature cultures grown on 24-well cell culture plates, wash the cells with phosphate-buffered saline (PBS) once and then add 500 µL of 0.25% trypsin-EDTA (Table of Materials). Incubate for 10-40 min in a cell culture incubator, checking every 5-10 min until the cells are rounded and almost ready to detach.
   2. Gently pipette media over the cells to detach them from the cell culture plastic, followed by transfer to 3x volume of cell culture media (1,500 µL per 24-well), spinning down in a centrifuge at 250 × g for 5 min at room temperature.
      NOTE: The recipe for RPE cell culture media is detailed in Table 1 and available in previously published references^{9,15,16,17,18}.
   3. Resuspend cells in RPE cell culture media with 15% fetal bovine serum (FBS) and count the cells with a hemocytometer.
   4. Seed 74,000 cells for each well of a 96-well plate (generally 225-300 × 10⁵ cells/cm²), after coating with highly specialized extracellular matrix coating substrates (Table of Materials) following the manufacturer's instructions.
      NOTE: Seed cells only on a Resipher-compatible plate (Table of Materials) and only in wells corresponding to the probe array on the sensing lid (columns 3, 4, 9, and 10 for the 32-channel lid; see Table of Materials). The system's sensing lid is available with 4-, 32-, or 96-channel sensors (see sensor locations in Figure 1A-D), and a range of standard cell culture plates are compatible.
   5. Since edge wells are prone to evaporation, which dramatically affects oxygen availability, and therefore, OCR readings, avoid seeding cells on all edge wells (Rows A and H of a 96-well plate). Furthermore, add 200 µL of sterile water in each of the empty wells of the 96-well plate to reduce evaporation effects.
   6. Keep the plate on a stable table surface for 10 min to allow the cells to settle; then return it to the incubator. Change the media after 24-72 h, replacing with standard RPE culture media with 5% FBS. Culture for at least 4 weeks, changing media 2x per week.
      NOTE: Cells are ready for experimentation when they are pigmented, cobblestone, and highly compact (Figure 1E).
2. Setting up and acquiring data with the system
   1. Place the sensing lid on an empty 96-well receiver plate and then place this back in the cell culture incubator. Align and mount the Device on the sensing lid and connect it to the Hub via the provided USB cable. This creates a Resipher "sandwich." (Figure 2A).
   2. Go to the Lucid lab website application (https://lab.lucidsci.com/) and click the New Experiment button on the right top corner to create a new experiment.
   3. Name the title of the experiment and input any relevant experimental notes for the particular study (e.g., passage number of the iPSC-RPE one is using).
   4. Create well conditions and treatment groups. For example, if testing the effects of different serum concentrations in the media on OCR, select Serum in group name, then enter media serum concentrations to be used, adding more serum percent values by clicking the + button. Assign which wells will receive a particular serum percentage and select a color pattern for those wells. Add more groups (e.g., a different experimental variable to test) as needed by clicking add group button.
   5. Define the plate setup and select the corresponding Device and plate style. Click add plate button and choose Device; select plate type. Select treatment and click on the corresponding well.
   6. Click the start now button to start the experiment. Check that the indicator on the website and the LED on the Hub is solid green.
   7. Allow the system to run for 15-60 minutes to ensure each sensor is properly calibrated and accurately detecting atmospheric oxygen, which should be at a concentration of ~200 µM in a fully humidified cell culture incubator with 5% CO₂ (Figure 2Bi).
      NOTE: If any of the sensors are more than 20% off from the average of the other sensors in a standard cell culture incubator setup, consider excluding that well in data analysis, as the sensor is faulty (Figure 2Bii).
      NOTE: If all of the sensors are more than 20% off from the average of the other sensors and from the expected atmospheric O₂ reported as 200 µM in a standard cell culture incubator setup, the sensing lid has been used too many times; replace it with a new sensing lid (Figure 2Biii).
      NOTE: Obtaining the O₂ readings in the air prior to starting an experiment significantly improves troubleshooting afterward. If a particular well appears as an outlier after an experiment is done and that well had a sensor that was out of calibration, the problem may be with the sensor, not the biological replicate.
   8. Remove the Device from the sensing lid (but do not detach the USB cable from the Device). Place the Device upside down in the incubator to allow the motor on the Device to reset. Do not reuse the Device until all the motor sounds have stopped (approximately 20-30 s).
      NOTE: It is important to keep the Device connected to the Hub via the USB cable at all times during an experiment, even when the Device is off the sensing lid. This allows the Device to continuously monitor and report on the cell culture incubator environment.
   9. Place the plate with the sensing lid back in the cell culture hood, along with a 96-well plate containing the RPE cultures. Change the media in the plate with RPE cultures with the same media and the same volume for all wells to obtain baseline OCR values for each well. Be sure to fill all the wells without RPE with sterile water to help prevent evaporation.
      NOTE: In general, we recommend 60-100 µL of RPE culture media per 96-well, based on data demonstrating that lower media volumes lead to rapid nutrient depletion but higher media volumes lead to inadvertent hypoxia⁹.
   10. Transfer the sensing lid to the plate containing cell cultures and the standard (non-Resipher) lid on the plate containing cell cultures to the empty 96-well receiver plate to maintain the sterility of the standard lid, which will be needed at various points during experimentation.
   11. Place the plate with cell cultures and the sensing lid back in the cell culture incubator. Once again, align and mount the Device on the sensing lid and connect it to the Hub via the provided USB cable (assembling the "sandwich"). Check that the indicators on the website interface and the Hub are all solid green.
       NOTE: O₂ concentration data will show up right away, while OCR data will only show up after enough O₂ data has been collected, approximately 1 h.
   12. Let the Device measure OCR on each well for at least 12-24 h to capture a baseline OCR.
       NOTE: Baseline OCR will differ depending on many factors, including the type of media being applied to cells. In Figure 2C, standard RPE culture media with different amounts of FBS (0%, 5%, 15%) have slightly different OCR values. Further, cultures with more FBS can sustain mitochondrial activity for longer after media is applied to cells before OCR drops due to depletion of mitochondrial metabolites (right side of curve).
   13. Once a baseline OCR is obtained, repeat steps 1.2.8-1.2.12, but change the media on the plate with RPE cultures to one's experimental conditions (typically, ± a drug or a comparison of different media conditions).
       NOTE: Routine media changes can also be handled the same way. Anytime the cell culture incubator is opened, expect significant disruption to OCR readings, as they are dependent on temperature, humidity, CO₂ concentrations, and other factors that are all temporarily disrupted with incubator door opening. During any removal of the Device from the sensing lid, there is no need to pause the experiment online. After the cell culture incubator door is opened or media is changed on the plate being probed, it will generally take 2-4 h to re-establish the oxygen gradient and begin measuring an accurate OCR.
   14. After data are obtained, subtract the baseline OCR for each well from the OCR after the experimental condition is applied, to determine the Delta OCR triggered by the treatment.
   15. After the experiment is completed, sterilize and reuse the sensing lid (which can be reused 3-5x, although performance degrades with repeated use). To sterilize the sensing lid, immerse the whole lid in 70% ethanol for 10 min in a cell culture hood, then remove from immersion and rest in cabinet probe side up (avoid touching the delicate probe tips), and let ethanol and water totally evaporate before putting the sensing lid on a new 96-well receiver plate.
3. Take brightfield images to normalize OCR values to cell number.
   ​NOTE: Melanin accumulation in RPE facilitates the normalization of OCR based on a simple count of cell numbers in living cultures using a brightfield microscope.
   1. Remove the Device and place it upside down, as in step 1.2.8. In the cell culture hood, swap the sensing lid on the RPE culture plate for a standard 96-well lid.
   2. Using a standard inverted microscope, take brightfield images of each well.
      NOTE: Keep the area one images consistent between wells (relative location in the well and objective magnification).
   3. In the cell culture hood, replace the standard 96-well lid on the RPE culture plate with the sensing lid and place it back in the incubator for further monitoring.
   4. Count the cell number in each well with ImageJ or other software.
   5. Normalize OCR to cell number in the different experimental groups. The system reports OCR in units of fmol∙(mm²)^-1∙s^-1-the consumption per cross-sectional unit area. Therefore, to normalize the OCR per cell, divide the OCR by cell count per mm² (rather than cell count per well). Equivalently, multiply the OCR reported by the system by the cross-sectional area of the well (around 31 mm² for a standard 96-well plate) to yield OCR in units of fmol∙s^-1∙well^-1.
4. Controls for determining RPE bioenergetic parameters.
   NOTE: To ensure the system and RPE cultures are responding to mitochondrial manipulations in predictable ways, one can test certain small molecules well-established to target particular steps of mitochondrial oxidative phosphorylation. These tests are analogous to the steps performed in a Mitochondrial Stress Test using the Seahorse Analyzer¹⁰ and provide a bioenergetic profile for RPE cultures.
   1. Culture RPE cells in 65 µL of standard RPE culture media with 5% FBS and measure OCR for 24 h to establish a baseline.
   2. Aspirate cultured media and add 65 µL of standard RPE culture media with 5% FBS and with mitochondrial uncouplers (3 µM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP] or 500 nM Bam15, Table of Materials). Incubate overnight.
      NOTE: Mitochondrial uncouplers should significantly increase OCR. Ensure the increased OCR is not at or near the theoretical maximum diffusion-limited OCR of approximately 275 fmol∙(mm²)^-1∙s^-1 for 65 µL of media in standard culture conditions. Max OCR for different commonly used media volumes is available in the Discussion section.
   3. After overnight incubation, aspirate the media with mitochondrial uncouplers and replace with 65 µL of standard RPE culture media with 5% FBS. Observe the plate for another 24 h to determine the recovery of RPE OCR after the removal of the drugs.
   4. Calculate the effects of uncouplers on OCR by subtracting the OCR obtained after treatment from the OCR obtained before treatment (Figure 3).
      NOTE: The increase in OCR with the addition of mitochondrial uncouplers signifies the reserve mitochondrial respiratory capacity of the cell¹⁹.
   5. To measure other mitochondrial parameters typically captured during the Mitochondrial Stress Test with a Seahorse Analyzer, perform the same steps above with the following compounds (Figure 4).
      1. Use oligomycin at 1 µM to assess for ATP-linked respiration.
      2. Use antimycin A and rotenone at 1 µM to assess for any non-mitochondrial respiration.
         NOTE: FCCP, oligomycin, antimycin A, and rotenone can be toxic to RPE. If they induce significant cell death, then OCR measurements will not be accurate. Thus, OCR measurements should be obtained only at a time point where there is no obvious cell death, generally within the first several hours after the drugs are added (Figure 4).

2. Measuring changes in mitochondrial metabolism in RPE undergoing EMT

1. Follow the same protocol as outlined in section 1.2.
2. To induce EMT after obtaining baseline OCR measurements, add TGF-β2 (10 ng/mL) to RPE cultures and monitor OCR over 3 weeks. Refresh media with fresh TGFβ2 every 2-3 days (Figure 5).
3. At least 2x a week, obtain brightfield images as in section 1.3 to ensure that the cell count is not changing. If the count does change, normalize the OCR values to the cell number in each well.
   NOTE: The same protocol can be used for other inducers of EMT to monitor long-term OCR changes throughout the culture period whilst the cells are in the incubator.

3. Measuring changes in mitochondrial metabolism in hypoxic RPE

NOTE: The application of the system under hypoxic, normoxic, or hyperoxic conditions is the same as section 1.2, except for keeping the "sandwich" in a hypoxia chamber (Table of Materials) placed in a standard cell culture incubator.

1. Drill a hole on the lid of the hypoxia chamber to mount the USB type-c cable through and seal it with silicone or putty (Figure 6A).
2. Assemble the sensing lid with the cell culture plate in the hood and transfer them into the hypoxia chamber.
3. Set up the "sandwich" (plate, sensing lid, Device) in the hypoxia chamber. Also place in the hypoxia chamber a Petri dish with sterile water to help maintain humidity. Finally, place a portable O₂ sensor in the hypoxia chamber (Table of Materials). Setup is displayed in Figure 6A.
4. Seal the chamber and connect it to a gas cylinder with a hypoxic concentration of O₂ and standard cell culture concentration of CO₂ (e.g., 1% O₂ and 5% CO₂).
5. Slowly exchange the air in the hypoxia chamber with the 1% O₂/5% CO₂ air in the cylinder until the oxygen sensor shows the O₂ concentration desired, usually somewhere between 1% and 10%.
   NOTE: The portable O₂ sensor requires time to equilibrate, so gas from the cylinder should be added into the hypoxia chamber slowly.
6. Close the inlet and outlet valves of the hypoxia chamber and transfer the whole chamber into the incubator.
7. Set up and start the experiment as in section 1.2.
   NOTE: Pre-equilibrating media in the hypoxia chamber prior to adding to cells can decrease the time it takes for the cell monolayer to experience true hypoxia (Figure 6B).

4. Calculating the O₂ concentration at the RPE monolayer to determine if cells are in hypoxic, normoxic, or hyperoxic conditions

NOTE: The system measures the O₂ concentration between approximately 1 to 1.5 mm above the bottom of the well by default assuming a standard plate is used with the corresponding recommended sensing lid for monolayer culture (refer to https://lucidsci.com/docs/LucidScientific_Sensing_Lid_Selection_Guide.pdf). While the O₂ concentration at the cell monolayer is not directly measured, data from the system can be used to estimate O₂ concentration at the level of the RPE. Specifically, knowing that an oxygen gradient exists between the top of the media column, where O₂ is available, and the bottom of the media column, where O₂ is being consumed, Fick's Laws of Diffusion can be combined with the measured OCR rate to extrapolate O₂ levels at the cell monolayer. A calculator for this estimation is provided online: https://lucidsci.com/notes?entry=oxygen_diffusion (and in the form of an open-source interactive notebook at https://observablehq.com/@lucid/oxygen-diffusion-and-flux-in-cell-culture, source code of this calculator could be found at https://github.com/lucidsci/oxygen-diffusion-calculator).

1. Once the calculator is accessed, enter the media volume in microliters (e.g., 100 µL).
2. Enter the saturated oxygen concentration at the air-liquid interface (the top of the media column above cells). This value is ~185 µM at standard atmospheric pressure with 5% O₂ and 95% humidity.
   NOTE: This can also be determined from O₂ measurements by system probes in media-only wells (with no cells at the bottom of the well).
3. In the Flux input, enter the Flux/OCR reported by the system.
4. Based on these values, the steady-state oxygen concentration at each height in the media column is calculated and shown in the plot (Figure 7). The x-axis of the plot is the height in mm above the bottom of the well. The y-axis is the calculated O₂ concentration at that height (in µM).
5. The O₂ concentration at the bottom of the well (where the cells are) is reported in the line below the plot.
   NOTE: In general, it is estimated that RPE in vivo sees an O₂ concentration of 4-9%. Each 10 µM of O₂ at standard atmospheric conditions corresponds to approximately 1% O₂. Thus, normoxia at the level of cells is approximately 40-90 µM of O₂. The higher the media column, the lower the O₂ will be at the level of the RPE monolayer⁹. Thus, if cells appear to be in hypoxic conditions, media volume can be reduced. If cells appear to be in hyperoxic conditions, media volume can be increased.

Results

The "sandwich" setup for the Resipher experiment is demonstrated in Figure 2A. Sensing lids with 32 probes corresponding to columns 3, 4, 9, and 10 on the 96-well plate sit between the cell plate and the Device. After connecting to the Hub, the Device activates motors to move the sensing lid up and down, measuring O₂ concentration in the media column at a range of heights above the cell monolayer (typically 1-1.5 mm). The O₂ gradient is therefore continuously me...

Discussion

Mitochondrial metabolism of the RPE plays a critical role in the pathogenesis of common blinding retinal diseases, including AMD and PVR. Modeling RPE mitochondrial metabolism in vitro allows one to isolate its metabolic state from those of surrounding tissues, along with subjecting the tissue to different disease-simulating insults in a controlled manner. Such in vitro modeling of RPE mitochondrial metabolism has been facilitated by the advent of high-fidelity human primary and iPSC-RPE cultures that a...

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-EDTA	Gibco	#25-200-056
3,3',5-triiodo-L-thyronine sodium salt	Sigma	T5516
32-channel Resipher lid	Lucid Scientific	NS32-101A for Falcon
Antimycin A from streptomyces sp.	Sigma	A8674-25MG	Inhibitor of Complex III of the electron transport chain
BAM15	Sigma	SML1760-5MG	Uncoupling agent to increase mitochondrial respiration
DMSO, cell culture grade	Sigma-aldrich	D4540-100ML	Vehicle for reconstituting mitochondrial drugs
Extracellular matrix coating substrates: Synthemax II-SC	Corning	#3535	Extracellular matrix for hfRPE
Extracellular matrix coating substrates: Vitronectin	Gibco	A14700	Extracellular matrix for iPSC-RPE
FCCP	Sigma	C2920-10MG	Uncoupling agent to increase mitochondrial respiration
Fetal Bovine Serum (Bio-Techne S11550H)	Bio-Techne	S11550H
Hydrocortisone-Cyclodextrin	Sigma	H0396
Hypoxia chamber	Embrient Inc.	MIC-101
N1 Media Supplement	Sigma	N6530
Non-Essential Amino Acids Solution	Gibco	11140050
O2 sensor	Sensit technology or Forensics Detectors	P100 or FD-90A-O2
Penicillin-Streptomycin	Gibco	15140-122
Recombinant human TGFβ2	Peprotech	100-35B	Transforming growth factor beta-2 to induce epithelial-mesenchymal transition
Rotenone	Sigma	R8875-1G	Inhibitor of Complex I of the electron transport chain
System-compatible plate	Corning	#353072
Taurine	Sigma	T8691
αMEM (Alpha Modification of Eagle's Media)	Corning	15-012-CV