Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

1Department of Ophthalmology and Visual Sciences, Washington University in St. Louis
Published 5/06/2015
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Summary

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

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Vinberg, F., Kefalov, V. Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System. J. Vis. Exp. (99), e52855, doi:10.3791/52855 (2015).

Abstract

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Introduction

Electroretinogram (ERG) is a well-established technique that can be used to record the electrical activity of the retina triggered by light. The ERG signal is generated mainly by voltage changes caused by radial currents (along the axis of photoreceptors and bipolar cells) flowing in the resistive extracellular space of the retina. The first ERG signal was recorded in 1865 by Holmgren from the surface of a fish eye1. Einthoven and Jolly 19082 divided the ERG response to the onset of light into three different waves, called a-, b-, and c-waves, that are now known to reflect mainly the activity of photoreceptors, ON bipolar cells, and pigment epithelium cells, respectively3-8. ERG can be recorded from the eyes of anesthetized animals or humans (in vivo), from isolated eye preparation9, across isolated intact retina (ex vivo)3,10-15 or across specific retina layers with microelectrodes (local ERG)4,16. Of these, in vivo ERG is currently the most widely used method to assess retinal function. It is a noninvasive technique that can be used for diagnostic purposes or to follow the progression of retinal diseases in animals or patients. However, in vivo ERG recordings produce a complicated signal with several overlapping components, often contaminated by extraocular physiological noise (e.g., breathing and cardiac activity).

Local ERG can be used to record the signal across specific layers of the retina but it is the most invasive and has the lowest signal-to-noise ratio (SNR) as compared to the other ERG recording configurations. Local ERG is also technically demanding and requires expensive equipment (e.g., microscope and micromanipulators). Transretinal ERG from the intact, isolated retina (ex vivo ERG) offers a compromise between in vivo and local ERG methods allowing stable and high SNR recordings from intact retinas of animals or humans17. Recently, this method has been used successfully to study rod and cone photoreceptor function in mammalian, primate and human retinas18-20. In addition, due to absence of pigment epithelium in the ex vivo retina, the positive c-wave component of the ERG signal is removed and a prominent negative slow PIII component is revealed in the ex vivo recordings. The slow PIII component has been shown to originate from the activity of Müller glia cells in the retina21-23. Thus, ex vivo ERG method could also be used to study Müller cells in the intact retina. Several studies have also shown that ex vivo ERG recordings could be used to measure concentration of pharmacological agents around the retina24 and test the safety and efficacy of drugs25-27.

Multiple commercial in vivo systems are available and used in many laboratories that do not necessarily have extensive electrophysiology background. In contrast, ex vivo devices have not been available until recently17 and as a result only very few laboratories are currently taking advantage of this powerful technique. It would be beneficial to make ex vivo ERG recordings available to more laboratories in order to advance our knowledge about retinal physiology and pathology, and to develop new therapies for blinding diseases. We demonstrate here a simple and affordable ex vivo ERG device17 and show how it can be used in combination with several commercially available in vivo ERG systems to record rod- and cone-mediated signaling (a- and b-waves) and the function of Müller cells (slow PIII) from intact wild-type mouse retinas.

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Protocol

All experimental protocols were in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the institutional Animal Studies Committee at Washington University.

1. Setting Up Perfusion and Specimen Holder

  1. Prepare solution for retina perfusion fresh on the day of the experiment. Use distilled and deionized water. Use one of the following three solutions.
    1. Prepare Bicarbonate-containing Ames’ solution (1 L): 1 bottle of Ames’ media and 1.9 g of NaHCO3,
    2. Prepare Locke’s solution (in mM): 112.5 NaCl, 3.6 KCl, 2.4 MgCl2, 1.2 CaCl2, 10.0 HEPES, 20.0 NaHCO3, 3.0 Na succinate, 0.5 Na glutamate, 0.02 EDTA, and 10.0 glucose, 0.1% MEM vitamins and amino acids
    3. Prepare HEPES-buffered Ringer solution (in mM): 133.3 NaCl, 3.3 KCl, 2.0 MgCl2, 1.0 CaCl2, 10.0 glucose, 0.01 EDTA, 12.0 HEPES, pH adjusted to 7.5 with ~5.8 ml of 1 M NaOH, add 0.72 g/L Leibovitz culture medium L-15. Use 20 - 50 μM DL-AP4 and 50 – 100 μM BaCl2 to isolate the photoreceptor response with any perfusion media.
  2. Prepare solution for electrodes28 (in mM): 140.0 NaCl, 3.6 KCl, 2.4 MgCl2, 1.2 CaCl2, 3 HEPES, 0.01 EDTA and adjust pH to 7.4 – 7.5 with NaOH. Electrode solution can be stored at RT for several months.
  3. Prepare and test the specimen holder.
    1. Glue black/grey filter paper on top of the domes of the specimen holder’s bottom part (see Figure 1A). Spread two-component 5 min epoxy glue carefully around the edges of the flat tops of the domes. If necessary, do it under dissection microscope.
    2. Wait until the glue is almost dry (about 4 min) and press the filter paper on the domes by using a flat item. Glue filter paper at least one day before the experiment. The filter paper can be used multiple times but should be replaced after a month of recordings. Use 70% ethanol to clean domes carefully from any glue residue before installing the replacement paper.
    3. Fill the electrode channels with electrode solution trying to avoid any air bubbles and screw pellet electrodes enclosed with a threaded adapter into the electrode channels (see Figures 1A and B). Connect the top and bottom pieces of the specimen holder with four screws and fill the perfusion lines with electrode solution.
    4. Measure the resistance and voltage between the leads of each electrode pair using a multimeter (Figure 1B). Resistance should be below 100 Ωk and the voltage below 10 mV if the channels are bubble-free and the electrodes are in good conditions.
  4. Pour 400 - 700 ml of perfusion media in the glass bottle. Separate another 300 ml to be used in the dissection of the retinas and store it in a fridge. Set the perfusion tubing in the heat exchanger block and place the preheated block on the heating plate (see Figure 1D).
  5. Enclose the bottle with a cap that has connections to CO2/O2 gas (if Ames’ or Locke’s is used) and for perfusion tubing (see Figure 1D). Preheat the bottle with the media to 37 oC and place it on the heating plate or on the top of the light stimulation unit in water bath set to 37 – 39 oC17.
    1. Prime the perfusion lines by filling them with perfusion media to initiate the gravity-driven flow.
      1. In LKC system, place the bottle on the top of the stimulator unit17 to provide a significant gravitational flow that is then adjusted by flow-rate regulators without being affected by the lowering level of the perfusion solution in the bottle during the experiment.
      2. In Ocuscience system, place the perfusion bottle inside the Faraday cage in order to minimize noise and place long perfusion output lines from the specimen holder (see step 2.8) well below the level of the specimen holder (and perfusion bottle) to increase gravitational drive of the solution. Shield these output lines and connect the shield to the amplifier’s ground to prevent coupling of the electromagnetic noise to the ERG signal.
  6. Adjust the perfusion to 3 – 5 ml/min by using flow-rate regulators. Connect 5% CO2 / 95% O2 gas from a cylinder with proper regulator and adjust the flow rate to ensure steady bubbling of the media in the bottle through an air stone.

2. Sample Preparation

  1. Assemble clean and sharp dissection instruments including straight-bladed microscissors, one or two 45o tweezers, razor blade and a rectangular piece of filter paper.
  2. Pour about 200 ml of cold perfusion solution in a large Petri dish so that the whole bottom part of the specimen holder (including the domes) can be immersed into the solution. This step becomes important when mounting the retinas on the domes (see step 2.6). Although some solutions are designed to be saturated with carbogen (5% CO2 / 95% O2), this is not essential for the dissection purposes and was not done in the experiments described here.
  3. For a typical experiment, keep animals in 12/12 hrs dark/light cycle and dark-adapt them for 6 – 12 hrs before the recordings. Euthanize the animal by CO2 inhalation followed by cervical dislocation under dim red light and do all the following procedures under dim red or IR light (use e.g., red filter in front of the microscope light source).
    1. Pull the eyes out by using tweezers and put them in the media. Place one eye at a time on a small piece of paper (e.g., some regular filter paper) and make a slit approximately on the level of ora serrata while holding the eye with tweezers (this is done outside of the solution here).
  4. Cut along the ora serrata (or closer to the equator of the eye) with microscissors and remove the cornea and the lens. Place the eye cup in the cold media in the large Petri dish and repeat the same procedure with the other eye.
  5. Cut a small incision from the top of the eye cup towards the optic nerve by keeping the scissors between the retina and sclera in order to keep the retina as intact as possible. Grip the sclera from both sides of the incision by using two tweezers and pull the tweezers away from each other to detach the retina.
    1. Cut the optic nerve and try to isolate the retina with a minimum amount of physical contact to the distal surface. RPE will mostly detach automatically from the retina during the dissection process. It is more important to avoid mechanical disturbance of the retina than to perform the dissection quickly, generally retinas can be incubated at least 30 min in the solution without significant effects on the response properties.
  6. Mount the retinas on the domes of the specimen holder (Figure 1C). Immerse the bottom part of the specimen holder in the Petri dish with the dissected retinas. Slide the retina, photoreceptor side (the convex surface of the isolated retina) upwards, above the dome and lift the specimen holder so that the retina attaches on the filter paper. Repeat the procedure for the other retina.
  7. Dry the holder plate carefully to prevent electrical crosstalk between the retinas as well as noise and signal shunting. Specimen holder has O-rings around the bottom part domes to prevent solution spill between the bottom and top parts of the holder as well as to help electrical isolation of the photoreceptor and ganglion cell sides of the individual retinas. Attach the top part of the holder with the four screws (see Figure 1B) and fill the perfusion channels with perfusion solution by using a syringe and a needle.
  8. Transfer the specimen holder next to the heat exchanger block and connect the input and output perfusion lines to the specimen holder (Figure 1D). Connect the electrodes to the ERG amplifier (top electrodes connect to the ground/minus pin in the amplifier) and attach the stimulator/control unit on the specimen holder by using an adaptor or slide the heating pad into the stimulator unit depending on which in vivo ERG system is used.

3. Recordings

  1. Configure the amplifier and stimulus settings by using the in vivo system’s software. Set the acquisition frequency to a value between 1 and 10 kHz and low-pass filtering to 300 Hz. Do not use high-pass filtering. Use 60 Hz (or 50 Hz in Europe) notch filter if necessary.
  2. Record baseline without light stimulation and with dim stimulation (e.g., green light of -35 dB or 0.3 mCd sm-2) to test for good electrical connection between the electrodes and retina sample. Wait 10 – 20 min before starting the data collection so that retinal temperature and function reach a stable state.
    1. Choose stimulus parameters according to any specific experiment and start data collection. E.g., use green light from -40 up to 0 dB or from 0.1 up to 1,000 mCd sm-2 for a scotopic response family. Use about 2 to 3 log-units brighter flashes in photopic recordings where rods have to be suppressed by background light (3 – 30 Cdm-2) or a probe flash (about 1.3 Cd sm-2 = 1 dB, see Figure 3).
    2. However, remember that the exact light intensity values might be somewhat dependent on your system calibration and experimental conditions. As a rule of thumb, scale intensities down by 5 – 10-fold as compared to in vivo recordings when using green light17. A black cover with apertures above the retinas (included in the commercial adapter system) can be used to reduce light scattering in the specimen holder and facilitate homogenous and equal stimulation of both retinas by the Ganzfeld sphere of the commercial in vivo systems.
      NOTE: For recordings from dark-adapted retina, the intensities of test flashes used in a typical experiment bleach only a negligible fraction of the pigment so that the lack of RPE-driven regeneration is not an issue. In addition, it has been previously shown that cone pigment regeneration can still take place in the isolated retina via the Müller cell visual cycle20.
  3. As experiments last typically from 30 min up to several hours, monitor baseline drift, noise level and response stability during the experiment as changes in these might indicate technical problems.
    ​NOTE: Increased noise or decreased response amplitudes may indicate air bubbles in the perfusion or electrode channels, too low or high temperature, solution leak/spill or displacement of the retina.

4. Cleaning

  1. Detach the specimen holder from the perfusion lines, open it and flush the retinas from the filter paper. Remove the electrodes and rinse them with distilled water (do not use ethanol). Clean the specimen holder (including the perfusion channels) with ethanol and/or distilled water. Flush perfusion tubing carefully with >70% ethanol.
  2. Rinse the perfusion bottle with distilled water (do not use detergents). Ethanol can be also used to clean the bottle.

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Representative Results

We recorded flash responses from dark-adapted wild-type (WT) C57BL/6 mouse retinas by following the experimental protocols described above and illustrated in Figure 1 by using different standard perfusion solutions (Figure 2). The response waveforms and kinetics as well as sensitivity of rod photoreceptors appeared similar in Ames’ and Locke’s media (Figure 2A and B). On the other hand, under HEPES-buffered Ringer solution (no bicarbonate or 5% CO2 / 95% O2) the response amplitudes were significantly smaller. We also found that in these conditions b-wave stability was compromised. Adding 40 μM APB (DL-AP4) removed positive b-wave efficiently in all three media (Figures 2D-F). Removal of the b-wave revealed a large slow negative wave (Figure 2D) that has been attributed to Müller cell activity21. Adding 100 μM of barium abolished this component, revealing the photoreceptor response of the ex vivo ERG signal (Figures 2D-F). We could record up to 1 mV saturated photoreceptor responses in Ames’ and Locke’s whereas maximum responses were typically around 200 μV under Ringer perfusion.

Cone photoreceptor responses have been isolated previously by so-called double flash technique where a bright probe flash saturating the rods is followed by a test flash at a time when cones have restored their dark-adapted state but rods remain saturated19,29. Here we isolated cone-mediated ERG responses (containing both a- and b-wave) in Ames’ media supplemented with 100 μM of barium but not DL-AP4 by using double flash technique (Figure 3). Barium was used to remove the slow glial component that appeared to make the late part of the responses more variable especially during repetitive use of bright flashes. We used a constant probe flash to saturate rods and variable test flashes 300 msec after the probe flash to elicit cone responses. A cone response family obtained by subtracting probe flash response from the ‘probe + test flash’ responses is shown in Figure 3B.

Figure 1
Figure 1. Use of ex vivo ERG specimen holder. (A) Filling of electrode channels and mounting of the electrodes. (B) Testing of the assembled specimen holder before dissection by measuring the resistance and voltage between the electrode pairs. (C) Mounting of the retina on the filter paper in the specimen holder. (D) The specimen holder connected to the perfusion lines and ERG amplifier in the commercial ERG system. The perfusion flow path is indicated by blue arrows. Please click here to view a larger version of this figure.

Figure 2
Figure 2. Flash response families recorded from dark-adapted WT mouse retinas perfused with Locke’s (A), Ames’ (B), and HEPES-buffered Ringer (C) medium. The flashes delivered 3, 40, 130, 390 and 1,400 photons μm-2 (from -36 to -9 dB or -3.6 to -0.9 log(Cd sm-2) green light (530 nm)). Black traces in (D-F) show responses recorded from retinas in (A-C) after addition of 40 μM APB and 100 μM barium. Red traces in (D) show responses recorded from the retina in (A) perfused with Locke’s supplemented with 40 μM APB but not barium. The inset shows the responses to the three dimmest flashes in Locke’s media containing APB and barium. The flashes ranged from 7 to 14,000 photons μm-2 (from -32 to 1 dB green light) in (D), from 3 to 1,400 photons μm-2 (from -36 to -9 dB green light) in (E), and from 7 to 1,400 photons μm-2 (from -32 to -9 dB green light) in (F). See Vinberg et al. 201417 and Lyubarsky et al. 199930 and 200431 for details of converting photopic luminous energy given in Cd sm-2 to photons μm-2. Please click here to view a larger version of this figure.

Figure 3
Figure 3. Isolation of cone responses with double flash method in WT mouse. (A) A response to probe flash (14,000 photons μm-2 or 1 dB green light, black) and a response to probe flash followed by a test flash (81,000 photons μm-2 or 9 dB green light, red). (B) Cone flash responses to test flashes ranging from 360 to 81,000 photons μm-2 (-14 to 10 dB green light) isolated by subtracting the probe flash response from the “probe + test flash” response. Please click here to view a larger version of this figure.

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Discussion

We demonstrate here the critical steps for obtaining high-quality ex vivo ERG recordings simultaneously from two isolated mouse retinas by using in vivo ERG system components together with an ex vivo ERG adapter. In this study we perfused both retinas from the animal with the same solution (either Ames’, Locke’s or Ringer) but it is also possible to perfuse each retina with a different solution e.g., for drug testing purposes. The most important steps for obtaining high quality data are shielding from electromagnetic noise, careful dissection of the retina, steady and relatively rapid perfusion flow by using an advanced custom-built specimen holder, and performing all sample preparation procedures under dim red (or IR) light. The method described here allows immediate use of both retinas and dual use of an in vivo ERG setup to perform in vivo and ex vivo ERG recordings.

The custom-built ex vivo ERG specimen holder recently designed by us17, and now commercially available, improves SNR by efficient electrical isolation of the proximal and distal parts of the retina and optimizes perfusion flow above the retina (high solution exchange rate). Absence of the slow frequency noise components in ex vivo ERG signal allows quantitative analysis even from very small responses. However, we found that ex vivo recording is more prone to interference of AC-power line noise (60 Hz in the US; 50 Hz in Europe) than in vivo experiments. This noise coupled to the signal mainly through the perfusion line, and it could be mostly removed by shielding (and grounding) all perfusion components (bottle, tubing) residing outside of the Faraday cage or Ganzfeld sphere. In addition, sometimes 60 Hz noise coupled to the ex vivo ERG signal through the heat exchanger and this noise could also be removed by grounding.

We demonstrate how to remove specific ERG signal components by adding pharmacological blockers into the perfusion during the experiment allowing dissection of the function of different cell types in the same retina/experiment with three different physiological perfusion media (Figure 2). A recent study showed that the choice of perfusion media affects photoreceptor physiology in single cell recordings32. Here we used Ames’, Locke’s and ‘HEPES-Ringer’ media to record dark-adapted flash responses in the absence and presence of pharmacological reagents intended to isolate the photoreceptor component of the ERG signal (Figure 2). Bicarbonate-buffered solutions gave larger a- and b-wave amplitudes, up to 1 mV. Photoreceptor dim flash responses under Locke’s medium with blockers contained complicated recovery waveform (see inset of Figure 2D) that was not seen with Ames’ or Ringer perfusion. When the use of ex vivo ERG becomes adapted by more laboratories it would be helpful to use the same perfusion media and standard methods to isolate different signal components. At this point it seems that the most versatile option is the Ames’ medium because it gives stable and large a- and b-wave amplitudes. In addition, the photoreceptor response, isolated pharmacologically in this solution, appears to have a simple waveform reminiscent of that recorded from single photoreceptors (Figure 2E). Yet, some open questions remain about the existence of other ERG signal components observed under in vivo conditions. For example, in our ex vivo recording conditions we did not see prominent oscillatory potentials, 100 – 150 Hz oscillating waves that are typically observed in the rising phase of the b-wave of an in vivo ERG response. It is thus possible that the inner retina function in our ex vivo conditions was compromised although large ex vivo b-waves implicated viable ON bipolar cell function. Future studies should resolve whether modifications in the experimental protocols shown here (dissection, perfusion etc.) would allow us to record oscillatory potentials under ex vivo conditions.

Cone function is vital for our vision. However, investigation of cones is hampered by their small size and scarcity especially in the mouse retina33. Isolation of cone function is further complicated in mouse ERG recordings because their M-cones and rods have almost identical spectral sensitivities30. Standard protocols take advantage of the several log-unit difference between rod and cone sensitivities by using rod-suppressing background light. However, steady background light is known to desensitize rods34,35 and affect cone function possibly through modulation of gap junctional coupling between rods and cones36,37. Thus, it is hard to find a background light intensity that is bright enough to keep rods saturated without affecting cones. Here we demonstrate an alternative double flash method that takes advantage of both the lower sensitivity and faster recovery kinetics of cones19,29,30. In this way it is easier to isolate truly dark-adapted cone-mediated responses. We noticed that in Ames’ or Locke’s solutions without any blockers, the details of the recovery waveform were somewhat affected during the course of the experiment by the use of bright probe and test flashes. This complicated the subtraction analysis to isolate the cone responses. However, removing the glial component by barium helped to stabilize the tail of the responses indicating that the variability was due to the Müller cell component. In this way it was possible to obtain dark-adapted cone-driven responses in WT mice (Figure 3). Cone responses isolated by double flash technique from WT mice appeared smaller as compared to those recorded from WT mice by using background light to suppress rod activity17,37. This difference can be explained by a well-characterized effect of background light that slowly (within 10 min) enhances cone response amplitudes probably due to removal of the rod-mediated suppression of cone responses37-39.

In summary, the method demonstrated here makes possible ex vivo electrophysiological recordings to study the function of the retina. In the future, we hope that many more laboratories will adapt this powerful method to study the physiology and pathology of animal and human retina and to advance our understanding of retinal function and develop better therapies for blinding diseases.

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Disclosures

Washington University in St. Louis has a license agreement with Xenotec, Inc. and may receive a royalty from the sale of the ex vivo adapter.

Acknowledgements

This work was supported by NIH grants EY019312 and EY021126 (VJK), EY002687 to the Department of Ophthalmology and Visual Sciences at Washington University, and by Research to Prevent Blindness.

Materials

Name Company Catalog Number Comments
In vivo ERG system OcuScience HMsERG www.ocuscience.us/id77.html
In vivo ERG system LKC Technologies UTAS-E 3000 www.lkc.com/products/UTAS/bigshot.html
Ex vivo adapter OcuScience Ex VIVO ERG adapter www.ocuscience.us/id107.html
Dissection microscope North Central Instruments Leica M80 May use any brand
IR emitter Opto Diode Corp. OD-50L www.optodiode.com
Prowler Night Vision Scopes B.E. Meyers Electro Optics D4300-I Military grade product.
Red filter Rosco Laboratories Roscolux #27 Medium Red May be used instead of IR system
Red head light OcuScience ERGX011 www.ocuscience.us/catalog/i29.html
Microscissors WPI, Inc. 500086 www.wpiinc.com/
Dumont tweezers #5 WPI, Inc. 14101
Razor blades Electron Microscopy Sciences 72000 www.emsdiasum.com
Scale Metler Toledo AB54-S/FACT May use any brand
pH meter and electrode Beckman Coulter pHI 350 May use any brand
NaCl Sigma-Aldrich S7653 May use any brand
KCl Sigma-Aldrich 60129 May use any brand
MgCl2 Sigma-Aldrich 63020 1.0 M solution
CaCl2 Sigma-Aldrich 21114 1.0 M solution
EDTA Sigma-Aldrich 431788 May use any brand
HEPES Sigma-Aldrich H3375 May use any brand
Sodium Bicarbonate Sigma-Aldrich S6297 May use any brand
Ames medium Sigma-Aldrich A1420 May use any brand
BaCl2 Sigma-Aldrich B0750 May use any brand
DL-AP4 Tocris Bioscience 101 May use any brand
Succinic acid disodium salt Sigma-Aldrich 224731 May use any brand
L-Glutamic acid Sigma-Aldrich G2834 May use any brand
D-(+)-Glucose Sigma-Aldrich G7528 May use any brand
Leibovitz culture medium L-15 Sigma-Aldrich L4386 May use any brand
MEM vitamins Sigma-Aldrich M6895
MEM amino acids Sigma-Aldrich M5550
Carbogen Airgas UN3156 5% CO2

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References

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