Quantitative In-Cell Hydrogen NMR Spectroscopy to Monitor Protein-Ligand Interactions

1. Reagent and solution setup

1. To prepare complete DMEM, add 5 mL L-glutamine 200 mM, 5 mL penicillin-streptomycin 100x, and 50 mL fetal bovine serum (FBS, 10% vol/vol final concentration) to 440 mL DMEM.     
   NOTE: This solution can be stored at 4 °C for 1 month.
2. Prepare agarose solution by dissolving 150 mg of low-gelling agarose in 10 mL of phosphate-buffered saline (PBS) at 85 °C to obtain a 1.5% (w/v) solution. Sterilize by filtration with a 0.22 µm filter. Prepare 1 mL aliquots of agarose solution in 1.5 N
3. Prepare the bioreactor medium.
   1. Dissolve 13.4 g of DMEM powder in 1 L of ultrapure H₂O.         
      NOTE: Depending on the application, the required final volume may differ (e.g., for 500 mL medium, dissolve 6.7 g of powder in 500 mL H₂O).
   2. Add 2% FBS, 10 mM NaHCO₃, 1x penicillin-streptomycin (100x), and 2% D₂O (e.g., for 500 mL medium, add 10 mL of FBS, 0.4 g of NaHCO₃, 5 mL of penicillin-streptomycin 100x, and 10 mL of D₂O).
   3. Measure the pH using a pH meter and if needed adjust to 7.4 by adding HCl.
      NOTE: Typically, the initial pH is very close to 7.4.
   4. Filter the bioreactor medium with a vacuum-driven sterile filter in a sterile 250 mL or 500 mL glass bottle.
   5. In the laminar flow hood, seal the bottle with a sterile steel headpiece with two hose nozzles and connect them to an FEP tubing (o.d. = 1/8", i.d. = 1.6 mm) that will be connected to the pump and to a 0.22 µm PTFE syringe filter for air intake.

2. Bioreactor setup

1. Assemble the flow unit using a second flow unit NMR tube, which will be later replaced with the one containing the cells. Refer to the flow unit operating instructions for the correct assembly.
   NOTE: At this point, the flow unit should be already cleaned (if not, perform step 4.2).
2. Set the water bath connected to the flow unit temperature control to 37 °C. Place the reservoir bottle in the water bath.
3. Connect the FEP tubing of the reservoir bottle to the pump.
4. Turn the bioreactor valve to "bypass" and prefill the pump with a medium.
5. Turn the bioreactor valve to "flow" and prefill the bioreactor with the medium at 0.1 mL/min.

3. Preparation of the cell sample

1. Collect the cells from the CO₂ incubator.
   1. Take a T75 flask of transfected HEK293T cells from the CO₂ incubator and remove the spent medium.
   2. Wash the cells twice with 7 mL (each) of PBS at room temperature (~20 °C).
   3. Use 2 mL of trypsin/EDTA to detach cells. After adding the solution, incubate for 5 min at room temperature to detach the cells.
      NOTE: Transfected cells may take slightly longer to get detached. If necessary, incubate the cells at 37 °C.
   4. Inactivate trypsin with 20 mL of complete DMEM; thoroughly resuspend the cells by pipetting up and down and transferring them in a 50 mL centrifuge tube.
   5. Centrifuge the cells at 800 x g for 5 min at room temperature and discard the supernatant.
   6. Wash the cells with 10 mL of PBS at room temperature to remove the residual medium.
   7. Centrifuge the cells at 800 x g for 5 min at room temperature and discard the supernatant.
   8. Transfer the cell pellet to a 1.5 mL capped microcentrifuge tube.
2. Embed cells in agarose threads.
   1. Melt one aliquot of solidified agarose at 85 °C in a water bath and subsequently keep it in solution at 37 °C in a block heater.
   2. With a Pasteur pipette, fill the bottom of the flow unit NMR tube with 60-70 µL of 1.5% agarose gel and place it in ice. This will create a ~5 mm high bottom plug that allows placing the cell sample within the active volume of the ¹H NMR coil.
   3. Heat up the pellet of cells obtained in step 3.1.8 at 37 °C for 15−20 s in the thermoblock.
   4. Resuspend cells in 450 µL of agarose solution. Be careful to avoid the formation of bubbles.
   5. Aspirate the cell-agarose suspension into a ~30 cm long chromatography PEEK tubing (i.d. = 0.75 mm) connected to a 1 mL syringe.   
      NOTE: Before aspiration, the tubing and the dead volume of the syringe should be prefilled with PBS at room temperature to avoid the formation of bubbles. The length of the tubing is not critical.
   6. Let the tubing cool down at room temperature for 2 min.
   7. Prefill the flow unit NMR tube with 100 µL of PBS at room temperature.
   8. Cast threads of cells embedded in agarose into the flow unit NMR tube by gently pushing the syringe.   
      NOTE: To fill the NMR tube homogenously, start by placing the end of the PEEK tubing at the bottom of the NMR tube and proceed towards the top while slowly swinging left-right.
   9. Repeat steps 3.2.5, 3.2.6, and 3.2.8 until all the cell-agarose suspension has been cast.
3. Insert cells in the bioreactor.
   1. Remove the empty NMR tube from the flow unit and increase the flow rate to 2 mL/min for a few minutes to remove residual gas bubbles in the inlet tubing.
   2. Set the flow rate to 0.2 mL/min and insert the NMR tube containing the cells by pushing it upwards slowly but steadily.       
      NOTE: The active flow of the medium avoids the backflow of tube content through the inlet that would otherwise occur during the insertion.

4. Bioreactor operation and cleaning

1. Bioreactor operation during the NMR experiment.
   1. Set the temperature in the NMR spectrometer to 310 K.
   2. Insert the flow unit in the spectrometer.
   3. Supply the bioreactor medium at a flow rate of 0.1 mL/min for the whole duration of the in-cell NMR experiments.
   4. At the desired time during the experiment, inject a concentrated solution of the external molecule into the medium reservoir bottle by piercing the silicone tubing with a sterile long-needle syringe.      
      NOTE: The final concentration of the molecule in the medium should be chosen based on previous knowledge of cell toxicity and, if available, on the predicted/estimated diffusion rate through the cell membrane.
   5. At the end of the NMR experiment, replace the tube containing the cells with an empty tube and rinse the flow unit with water.
2. Bioreactor clean-in-place.
   1. Clean the flow unit by flowing the following solutions at 1 mL/min: 0.2 M sodium hydroxide (NaOH); 3 M citric acid; 0.2 M NaOH, for at least 30 min each, followed by sterile-filtered ultrapure water for >2 h.
   2. Clean and autoclave the reservoir bottle and tubing assembly after each run.

5. NMR experiments

1. Setup of the NMR experiments.
   NOTE: Perform these steps beforehand, prior to the preparation of the in-cell NMR sample, to avoid any delays between cell collection and data acquisition.
   1. Create a new dataset at the NMR spectrometer and set the parameters for the desired NMR experiments.
   2. Set parameters for 1D ¹H NMR experiments.
   3. Center the ¹H carrier frequency at 4.7 ppm on the water signal.
   4. Select the zgesgp pulse program, set the spectral width to 20 ppm, and a 1,000-µs 180° square pulse for water suppression. Set an inter-scan delay of 1 s. Acquire the spectrum with 32 scans.
   5. For cells expressing unlabeled CA II: select the p3919gp pulse program, set the spectral width to 30 ppm to cover the imino region of the spectrum, and adjust the delay for binomial water suppression so that the maximum excitation is centered at the chemical shifts of the signals of interest (d7 = 20 µs at 950 MHz). Set an inter-scan delay of ≥1 s. Acquire with 512 scans.
   6. For cells expressing ¹⁵N-labeled SOD1: select the sfhmqf3gpph pulse program, set the ¹H and ¹⁵N spectral widths to 16 and 50 ppm, respectively, the shaped pulse offset and excitation bandwidth to 8.5 and 6 ppm, respectively, and a 350 µs pulse for decoupling scheme (garp4 or other depending on the instrument). Set an inter-scan delay of 0.3 s. Acquire 16 scans and 128 increments in the ¹⁵N dimension.
2. Real-time NMR spectra acquisition.
   1. Once the bioreactor is inserted in the NMR spectrometer, wait for a few minutes to allow the exchange of the medium.      
      NOTE: This process is easily monitored from the appearance of the lock signal as the PBS is replaced with a medium containing 2% D₂O.
   2. Adjust the matching and tuning of the ¹H channel, shim the magnet, and calculate the ¹H 90° hard pulse length.
   3. Adjust the ¹H power levels in each pulse sequence according to the ¹H hard pulse.
   4. Record a first zgesgp ¹H spectrum to check the sample content and the field homogeneity.
   5. Copy the zgesgp and the p3919gp/sfhmqcf3gpph experiments to the desired number and queue them in the acquisition spooler. 
      ca: The zgesgp spectra are only used to control the state of the sample and the field homogeneity; therefore, they can be either skipped or recorded less frequently.
   6. For cells expressing unlabeled CA II: process the p3919gp spectra by applying zero filling and exponential line broadening window function (LB = 20 Hz).
   7. For cells expressing ¹⁵N-labeled SOD1: process the sfhmqcf3gpph spectra by applying zero filling and squared sine bell window function (SSB = 2) in both dimensions.         
      NOTE: The size of the processed spectra can be further reduced by removing regions free of signals (in Topspin, this is done by setting the desired STSR and STSI values).