Key result
Cryogenic transfer protocol for MAS NMR preserves cellular viability versus typical sample handling.
Why the study?
Freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR, resulting in loss of cellular integrity and viability.
This protocol enables in-cell structural biology on cryopreserved cells by preserving cellular viability during sample loading into a MAS NMR spectrometer.
Dynamic nuclear polarization (DNP) can dramatically increase the sensitivity of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy. These sensitivity gains increase as temperatures decrease and are large enough to enable the study of molecules at very low concentrations at the operating temperatures (~100 K) of most commercial DNP-equipped NMR spectrometers. This leads to the possibility of in-cell structural biology on cryopreserved cells for macromolecules at their endogenous levels in their native environments. However, the freezing rates required for cellular cryopreservation are exceeded during typical sample handling for DNP MAS NMR and this results in loss of cellular integrity and viability. This article describes a detailed protocol for the preparation and cryogenic transfer of a frozen sample of mammalian cells into a MAS NMR spectrometer.
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Ghosh et al. (2020) studied this question. Cryogenic sample loading protocol vs. Typical sample handling was evaluated on Cellular integrity and viability. A detailed protocol for the preparation and cryogenic transfer of frozen mammalian cells into a MAS NMR spectrometer preserves cellular viability compared to typical sample handling.
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