In Situ Detection of Endogenous HIV Activation by Dynamic Nuclear Polarization NMR and Flow Cytometry

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Date
2020-07Type
- Journal Article
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Cited 10 times in
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Cited 11 times in
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ETH Bibliography
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Abstract
We demonstrate for the first time in-cell dynamic nuclear polarization (DNP) in conjunction with flow cytometry sorting to address the cellular heterogeneity of in-cell samples. Utilizing a green fluorescent protein (GFP) reporter of HIV reactivation, we correlate increased 15N resonance intensity with cytokine-driven HIV reactivation in a human cell line model of HIV latency. As few as 10% GFP+ cells could be detected by DNP nuclear magnetic resonance (NMR). The inclusion of flow cytometric sorting of GFP+ cells prior to analysis by DNP-NMR further boosted signal detection through increased cellular homogeneity with respect to GFP expression. As few as 3.6 million 15N-labeled GFP+ cells could be readily detected with DNP-NMR. Importantly, cell sorting allowed for the comparison of cytokine-treated GFP+ and GFP− cells in a batch-consistent way. This provides an avenue for normalizing NMR spectral contributions from background cellular processes following treatment with cellular modulators. We also demonstrate the remarkable stability of AMUPol (a nitroxide biradical) in Jurkat T cells and achieved in-cell enhancements of 46 with 10 mM AMUPol, providing an excellent model system for further in-cell DNP-NMR studies. This represents an important contribution to improving in-cell methods for the study of endogenously expressed proteins by DNP-NMR. Show more
Permanent link
https://doi.org/10.3929/ethz-b-000425969Publication status
publishedExternal links
Journal / series
International Journal of Molecular SciencesVolume
Pages / Article No.
Publisher
MDPIOrganisational unit
09681 - Barnes, Alexander / Barnes, Alexander
09681 - Barnes, Alexander / Barnes, Alexander
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Citations
Cited 10 times in
Web of Science
Cited 11 times in
Scopus
ETH Bibliography
yes
Altmetrics