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

1. Plating H-RPE in the cell culture plate

1. 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 µg/mL amphotericin).
2. Incubate the cells at 37 °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.
3. 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).
   1. Rinse H-RPE gently with 3 mL of HEPES buffered saline. Then add 3 mL of trypsin/EDTA and incubate (37 °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.
   2. Centrifuge the cells at 200 x g for 3.5 min to form the cell pellet. Carefully aspirate and discard the supernatant.
4. Resuspend the cells in human RPE medium to a final concentration of 20,000 cells per 100 µL per well.
   1. 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.
   2. Be sure to leave the four corner wells empty of cells (100 µL 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.
5. Leave the cell culture plate at room temperature (RT) for 1 h before placing it back into the incubator (5% CO₂, 37 °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.
6. 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.

2. The day before running the assay

1. Ensure that the sensor cartridge is hydrated the day before the assay by filling each well of the utility plate with 200 µL 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.
2. 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 °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.
3. Ensure that the instrument is turned on and start the software to allow the instrument to stabilize to 37 °C overnight (Figure 2). Generally, the instrument can be left on even when not in use.

3. Real-time Mito Stress Test using the extracellular flux analyzer

1. 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.
2. 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 °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.
3. Remove the human RPE media and replace it with 180 µL of freshly prepared assay media (step 3.2). Place the cell culture plate in a humidified 37 °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.
4. 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 µM 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 µM. Pipette 20 µL of the 10x drug stock into Port A, 22 µL into Port B, and 25 µL into Port C, to achieve the specified final drug concentration in each well. For protocols requiring all four drug ports, pipette 28 µL 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.
5. Open the Templates tab in the analysis software, select Mito Stress Test, and fill out the Group Definitions.
   1. 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.
   2. 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.
6. 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.
   1. 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.
   2. 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).
7. 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.
8. 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 µL 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).
9. Wrap the edges of the plate in Parafilm to prevent evaporation and place it in a -80 °C freezer to aid in cell lysis overnight, before quantification of the protein content using the BCA assay.
10. 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.
    ​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.

4. Real-time Glycolytic Stress Test using the extracellular flux analyzer

1. 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.
2. 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.
   ​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.

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 higher temperatures (37 to 60 °C). If there are more peptide bonds, making the amount of Cu²⁺ proportional to the protein content in the solution¹⁶. This reaction results in a 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 µg/mL.

1. 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.
2. Pipette 25 µL of each BSA serial dilution in duplicate into the 96-well plate.
3. Pipette 25 µL of the 1x lysis buffer in duplicate into the 96-well plate to serve as the blank.
4. Thaw the cell culture plate to room temperature and pipette 25 µL of each cell lysate in duplicate into the 96-well plate.
5. 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 µL of the working reagent to each well.
6. Protect the plate from light by covering the plate in foil and incubate in a 37 °C oven for 20 min.
7. Measure the absorbance at 562 nm in a 96-well plate reader.
8. 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 µg/mL. Use the linear equation derived from the standard curve to determine the protein concentration of the experimental samples.