An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

All animal experiments were carried out in accordance with the LIG-4 animal protocol, which was approved by the NIAID Animal Care and Use Committee.

1. Preparation of Reagents

1. Prepare magnetic separation (MS) buffer: PBS (pH 7.2) supplemented with 0.5% BSA and 2 mM EDTA or automatic separation solution supplemented with 0.5% BSA. Sterile filter (0.22 µm) and store at 2-8 °C.
2. Prepare RF10 medium: RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin, 50 µM streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 µM 2-mercaptoethanol, 10 mM HEPES. Use sterile reagents. Sterile filter and store at 2-8 °C.
3. Prepare mitochondrial stress test medium: XF base medium supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine and 25 mM glucose.
4. Prepare glycolysis stress test medium: XF base medium supplemented with 2 mM L-glutamine.
5. Adjust the pH of both the mitochondrial and glycolysis stress test media to 7.4, sterile filter and store at 2-8 °C. Warm media to 37 °C and re-adjust the pH before use (carry out pH measurements at 37 °C).
   NOTE: Using XF medium, which lacks bicarbonate, is critical. Since the atmosphere in the extracellular flux analyzer contains only ambient CO₂, any bicarbonate in the medium, upon binding to protons released as a byproduct of glycolysis, will dissociate to form CO₂ and water. CO₂ will degas during the experiment, causing drifts in pH that would obscure the glycolytic acidification signal.
6. Prepare oligomycin: Prepare 10 mM stock solution in DMSO; store at -20 °C.
7. Prepare 2,4-DNP: Prepare 1 M stock solution in DMSO; store at -20 °C.
8. Prepare antimycin A: Prepare 10 mM stock solution in DMSO; store at -20 °C.
9. Prepare rotenone: Prepare 10 mM stock solution in DMSO; store at -20 °C.
10. Prepare glucose: Dissolve glucose in glycolysis stress test medium to form a 250 mM solution; prepare fresh before each experiment and do not freeze.
11. Prepare 2-DG: Dissolve solid 2-DG in glycolysis stress test medium to form a 500 mM solution; prepare fresh before each experiment and do not freeze.

2. Harvesting Spleen and Lymph Nodes from Mice

1. Euthanize the mouse by CO₂ asphyxiation followed by cervical dislocation.
2. Spray the mouse with 70% ethanol.
3. For subsequent T cell isolation, harvest the spleen, superficial cervical, superficial/deep axillary, mandibular, inguinal, and mesenteric lymph nodes.
4. For subsequent B cell isolation, harvest only the spleen.
   NOTE: Use a biosafety cabinet and keep the harvested organs in PBS or RF10 media on ice until processing. Use sterile labware and sterile technique for all processing and isolation steps. Keep all buffers and media on ice to increase isolation efficiency and to reduce cell death.

3. Tissue Processing

1. Place a 70 µm cell strainer over a 50 ml conical tube and transfer the organs onto the strainer. For T cell isolation, combine all the organs and for B cell isolation transfer only the spleen.
2. Using the plunger from a sterile 3 ml syringe, mash the tissue through the strainer. During mashing, make sure that the filter and the organs are moist at all times and wet them as required by adding MS buffer. This will both keep cell viability high and prevent clogging of the filter.
3. After mashing, apply 5-10 ml MS buffer to the filter to increase cell recovery. Centrifuge the suspension at 400 x g for 5 min at 4 °C. (Carry out all further centrifugations under these conditions, unless specified).
4. Decant the liquid and resuspend the pellet in 5 ml ACK red blood cell lysis buffer. Incubate for 5 min on ice to lyse red blood cells (red blood cells need to be removed because they interfere with the magnetic separation). Add 10-20 ml MS buffer and centrifuge.
5. Place a 30 µm cell filter over a 15 ml conical tube and prime it with 1 ml MS buffer (this filtration will remove debris from the erythrocyte lysis). Decant the liquid from the centrifuged tube, resuspend the pellet in 5 ml MS buffer and pass it through the filter. Wash the initial tube with 4 ml MS buffer to increase cell recovery, and use this to rinse the filter.
6. Using a hemocytometer or an automated cell counter, determine the total number of cells. This cell number will be used to determine the amount of magnetic separation reagents required in Section 4. If splenocytes are to be used in the subsequent extracellular flux assay, set aside the appropriate number of cells at this time.
7. Centrifuge the suspension and proceed to magnetic separation. Resuspend splenocytes not used for B cell isolation in 5 ml RF10 and keep on ice until the extracellular flux assay.

4. Magnetic Separation of B Cells and Naïve CD4⁺ T Cells

1. Determine the necessary volumes of biotin-antibody cocktail, anti-biotin microbeads, and MS buffer. Use the following volumes for 10⁸ cells as a guide and scale up or down as necessary.
   NOTE: Incubate samples in a refrigerator rather than on ice since incubating on ice may reduce antibody binding efficiency and longer incubation times might be required.

Table 1: T cell isolation volumes and instructions.

Table 2: B cell isolation volumes and instructions.

2. Resuspend the pellet in MS buffer: 500 µl for manual separation, 3-4 ml for automatic separation.
3. Continue with either manual or automated separations as follows:
   1. Manual Separation:
      1. Insert an LS column into the separation magnet, place a sterile 15 ml conical tube below to collect the flow through, and prime the column by washing with 3 ml MS buffer. Discard the flow through.
      2. Transfer the cell suspension to the primed LS column and allow it to pass through; wash the tube used during incubation with 3 ml MS buffer and pass this through the column as well. The eluate will contain the purified cells. For B cell isolation, pass the purified cells through the column a second time into a new 15 ml tube, and repeat the washing step. This will increase purity and yield.
   2. Automated Separation:
      1. Switch on the automatic separator and check the levels of running buffer, rinsing solution and 70% Ethanol. Ensure that the waste is empty before starting the separation.
      2. Select the appropriate chilled rack depending on the tube size of the unpurified sample (e.g., 15 ml). In order to keep the cells viable throughout the separation process, use racks that have been previously chilled at 2-8 °C for 3-4 hr, or until the coolant becomes solid.
      3. Mount the chilled rack on the sample stage of the automatic separator.
      4. Place the sample tube in slot A1, a tube for the negative fraction in slot B1, and a tube for the positive fraction in C1. If collecting more than one sample, place additional tubes in the next columns (A2, B2, C2, etc.).
      5. Select the separation tab on the touchscreen, and indicate the arrangement of tubes on the rack. From the separation menu, select the "Depletes" program and complete the program cycle with the appropriate type of wash (see note below).
         NOTE: The "Depletes" program carries out depletion using the machine's most sensitive mode, which prioritizes purity. Additionally, there are three different wash options: quick rinse, rinse, and sleep. If the samples are from the same source and are to be combined, select quick rinse. If the samples are to be kept separate, select rinse. Select the sleep option if no further isolations will be carried out that day and if the machine will be shut down following isolation
      6. Start the isolation by pressing "Run," and confirm the buffer levels when prompted. After the program has ended, the negative fraction will contain purified cells.
   3. Fill the conical tube up to 15 ml with MS buffer and centrifuge.
   4. Decant the MS buffer and resuspend cells in 5 ml RF10 media. Keep cells on ice until the extracellular flux assay.
      NOTE: Ideally, the extracellular flux assay should be carried out immediately after isolation. However, in preliminary experiments no significant differences in assay outcomes were observed in cells used within 3 hr of isolation versus freshly-isolated ones.

5. Extracellular Flux Assay

NOTE: The protocol outlined here is for the 96-well format of the instrument. Volumes will need to be adjusted if another format is used.

1. Hydration of Sensor Cartridge
   1. Lift up the sensor cartridge, fill each well of the utility plate with 200 µl of calibrant solution and lower the sensor cartridge onto the utility plate, submerging the sensors in the calibrant solution.
      NOTE: Verify that the calibrant solution level is high enough to keep the sensors submerged. The cartridge harbors fluorophores associated to O₂ and H⁺ for each well; these need to be hydrated in order for them to work properly.
   2. Place the cartridge in a 37 °C incubator not supplemented with CO₂ or oxygen/nitrogen. Carry out all further incubations under these conditions, unless specified. Incubate the cartridge overnight to hydrate.
2. Preparation of Adhesive-coated Plates
   1. Prepare 2.5 ml of a 22.4 µg/ml adhesive solution in 0.1 M sodium bicarbonate, pH 8.0 (bicarbonate provides the optimum pH for adhesive adsorption).
   2. Apply 25 µl of the solution to each well of the assay plate, and incubate on the bench at room temperature for 20 min.
   3. Aspirate adhesive and wash each well twice using 200 µl of sterile water. Wait until wells are dry before seeding the cells.
3. Seeding Cells in Adhesive-coated Plates
   1. Centrifuge cells at room temperature at 200 x g for 5 min. Resuspend cells in 5 ml of the appropriate assay medium (see above for formulation of mitochondrial stress and glucose stress media). Centrifuge cells again and resuspend at the desired concentration in assay medium (resuspension volume will vary based on concentration; each well will contain 180 µl of cell suspension).
      NOTE: As long as the appropriate assay media and compounds are used, the extracellular flux analyzer can simultaneously run the mitochondrial and glycolysis stress tests on the same plate.
   2. Plate 180 µl of cell suspension in each well. Use wells A1, A12, H1, and H12 for background temperature correction: add 180 µl of assay medium in these wells (no cells).
   3. Incubate plates for 25 min at 37 °C.
   4. Centrifuge the plate at 200 x g for 5 min with no brake to ensure that all cells have completely attached. Visually confirm that the cells are stably adhered to the culture surface, forming a monolayer, by viewing under the microscope. Transfer the plates back to the incubator for an additional 30 min.
4. Preparation of 10x Concentrations of Compounds, and Loading of Injector Ports
   1. For the mitochondrial stress test, prepare 2.5 ml each of 10 µM oligomycin, 1 mM DNP, and a mixture of 10 µM rotenone and 10 µM antimycin A, all in mitochondrial stress assay medium.
      NOTE: The concentration of 2,4-DNP is based on previously-published studies;²¹ this is a general guideline but the concentration of uncoupler for each cell type used should be determined by the researcher.
   2. For the glycolysis stress test, prepare 2.5 ml each of 250 mM glucose, 10 µM oligomycin, and 500 mM 2-deoxyglucose (2-DG) in glycolysis stress assay medium.
   3. Warm 10x solutions to 37 °C, and adjust pH to 7.4.
   4. Load the compounds into the appropriate injector ports of the cartridge using a multichannel micropipettor and the loading guides, as follows:

      Port	Mitochondrial Stress Assay	Glycolysis Stress Assay
      A	20 µl oligomycin	20 µl glucose
      B	22 µl 2,4-DNP	22 µl oligomycin
      C	25 µl antimycin A/rotenone	25 µl 2-DG

      Table 3: Compound volumes.
      
      NOTE: Each series of ports (e.g., all ports A) must contain the same volume. It is critical that all wells in a given series are loaded, even those not used in the experiment, otherwise the compounds will not be injected (ports in unused wells can be loaded with the same volume of assay medium).
   5. Incubate the cartridge while setting up the program.
5. Setting Up Extracellular Flux Assay Protocols
   1. Set up the following program (Table 4).

      Step	Loop
      Calibration	––
      Equilibration	––
      Baseline readings	3 times: Mix 3 min, wait 0 min, measure 3 min
      End loop	––
      Inject Port A	––
      Measurements	3 times: Mix 3 min, wait 0 min, measure 3 min
      End loop	––
      Inject Port B	––
      Measurements	3 times: Mix 3 min, wait 0 min, measure 3 min
      End loop	––
      Inject Port C	––
      Measurements	3 times: Mix 3 min, wait 0 min, measure 3 min
      End loop	––
      End Program	––

      Table 4: Program layout.
      NOTE: In certain conditions, e.g., when using activated cells, the acidification may continue for longer than in naïve cells. For this reason, it may be necessary to extend the "wait" times in the above program or repeat each measurement cycle more times.
   2. Begin the program. After the calibration step, replace the calibrant plate for the assay plate (when prompted).
      NOTE: Using the software, it is possible to indicate groups of wells that have similar conditions. Additionally, it is crucial to indicate which wells are the control wells (in this protocol, they are the four corners of the plate) and to indicate which wells are empty. For a more detailed, step-by-step, protocol for setting up the machine and its software, the manufacturer's website should be consulted.
6. Measuring Protein Content
   1. Remove the remaining assay medium from each well without disturbing the cells.
      NOTE: If it is not convenient to proceed with the protein concentration assay, it is possible to freeze the whole plate at -20 °C until analysis. If frozen, thaw before continuing.
   2. Prepare a 1x solution of protease inhibitors (100x stock) in RIPA lysis medium (sufficient for 50 µl/well).
   3. Add 50 µl RIPA lysis medium supplemented with protease inhibitors to each well. Agitate plate on a shaker for 5 min, and then incubate plate on ice for 30 min to fully lyse cells.
   4. Spin the plate at 200 x g for 5 min at room temperature to bring lipids and other molecules to the bottom of the plate so that they do not interfere with the bicinchoninic acid (BCA) assay.
   5. Measure protein concentration by BCA assay according to manufacturer's recommendations.