Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

Presented here is a protocol for cryogenic transfer of frozen samples into the dynamic nuclear polarization (DNP) magic angle spinning (MAS) nuclear magnetic resonance (NMR) probe. The protocol includes directions for rotor storage prior to the experiment and directions for viability measurements before and after the experiment.

Transcript

The sensitivity of DNP assisted MAS NMR spectroscopy allows us to characterize proteins inside intact cells at their endogenous concentration. This application requires careful sample handling. Some morphologies preserved and cells are viable when they are cryogenically transferred into the spectrometer using this protocol.

Introduction of the cold rotor can cause moisture and ice build up, which can mechanically prevent sample spinning up. This demonstration will guide experiments to perform the cryogenic transfer with confidence. To begin, place a cushion made from a piece of tissue or paper towel under the lid and at the bottom of the cryogenic vial.

Place the 3.2 millimeter sapphire rotor into the cryogenic vial padded with the tissue paper, with the marked end facing the bottom of the vial. Slow freeze the rotor by placing the vial into a controlled rate cooling container and place the container in a 80 degrees Celsius freezer for a minimum of three hours. Transfer the cryogenic vial containing the frozen rotor to a small vacuum flask filled with liquid nitrogen.

Use the vacuum flask to carry the sample to the spectrometer facility. Fill a dry, thermally insulated, wide mouth foam vacuum flask with 500 milliliters to one liter of liquid nitrogen. Take the cryogenic vial from the transfer flask and hold it just above the surface of the liquid nitrogen to protect it from the atmosphere.

While holding the cryogenic vial with the vial's mouth pointing slightly downwards, unscrew the cap, and let the rotor slide into the liquid nitrogen bath. Pre-chill a 1.5 milliliter microcentrifuge tube by submerging it in the liquid nitrogen. Under the surface of the liquid nitrogen bath, use tweezers to transfer the rotor into the microcentrifuge tube with the drive tip facing the bottom of the tube and the markings facing the opening.

Use tweezers to hold the tube containing the rotor under liquid nitrogen by its neck. With a second pair of tweezers, hold the NMR sample catcher above the surface of the liquid nitrogen inclined at an acute angle with respect to the microcentrifuge tube. To transfer the rotor from the NMR sample catcher to the NMR spectrometer, place the cryo cabinet in ejection mode by pressing eject.

Insert the open end of the NMR sample catcher into the microcentrifuge tube while still under the surface of the liquid nitrogen. Lift both the microcentrifuge tube and the NMR sample catcher to allow the rotor to fall into the sample catcher. Shake the sample catcher and the tube if the rotor gets stuck on the rim.

Leave the empty microcentrifuge tube on top of the NMR sample catcher to shield the rotor from air. Remove the other empty sample catcher from the probe and lay it on the floor. Transfer the NMR sample catcher containing the rotor to the other hand.

Then remove the microcentrifuge tube and insert it immediately into the probe. Signal the person operating the cryo cabinet to press stop eject and insert. Spin up the sample to the desired spinning rate by adjusting the bearing and driving flow pressure.

Pour 500 milliliters to one liter of liquid nitrogen into a wide mouth foam vacuum flask and place the bath under the spectrometer. Submerge the empty cryogenic vial containing a piece of tissue paper in the liquid nitrogen bath. Reduce the spinning rate to zero kilohertz by ramping down the driving and bearing gas flow, and eject the rotor by switching to the ejection mode.

While keeping the ejection mode on, remove the sample catcher from the probe and drop the rotor directly into the wide mouth foam flask containing liquid nitrogen. Using pre-chilled tweezers, transfer the rotor into the pre-chilled cryogenic vial under the surface of the liquid nitrogen. Pre-chill the cryogenic vial cap by dipping it into liquid nitrogen.

Remove the vial containing the rotor and liquid nitrogen from the bath, and cap the tube with the pre-chilled cap. Resubmerge the cryogenic vial in liquid nitrogen. The sample can be transferred to longer term liquid nitrogen storage or unpacked immediately for further analysis.

Cryogenic insertion of pre-frozen samples of mammalian cells into the NMR spectrometer supports viability throughout the NMR experiment. Using this protocol, the trypan blue permeability of mammalian cells after MAS NMR is similar to that of cells not exposed to any temperature change. If cells are slow frozen, then warmed to room temperature before insertion, cellular viability decreases to less than 10%Meanwhile, cryogenic insertion of frozen samples of mammalian cells support cell viability throughout the NMR experiment.

When attempting does protocol, request assistance with pressing eject and spinning the sample up so that the sample can be introduced into the probe before it warms up, making sure that no moisture is introduced into the probe well-assured that the spectrometer will run cold for many weeks. This protocol is applicable to any system where temperature fluctuations might compromised sample integrity, such as freeze quenched reactions, or characterization of trapped reaction intermediates enzymology, and protein folding.

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Journal (Biology)

Journal (Biology)

Cryogenic Sample Loading into a Magic Angle Spinning Nuclear Magnetic Resonance Spectrometer that Preserves Cellular Viability

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