TY - JOUR
T1 - High-resolution imaging of protein secretion at the single-cell level using plasmon-enhanced FluoroDOT assay
AU - Seth, Anushree
AU - Mittal, Ekansh
AU - Luan, Jingyi
AU - Kolla, Samhitha
AU - Mazer, Monty
AU - Joshi, Hemant
AU - Gupta, Rohit
AU - Rathi, Priya
AU - Wang, Zheyu
AU - Morrissey, Jeremiah J.
AU - Ernst, Joel D.
AU - Portal-Celhay, Cynthia
AU - Morley, Sharon Celeste
AU - Philips, Jennifer A.
AU - Singamaneni, Srikanth
N1 - Funding Information:
We thank Dr. Nathaniel Huebsch from the Department of Biomedical Engineering at Washington University in St. Louis for providing access to the epifluorescence microscope used in this study. The authors thank Nano Research Facility (NRF) and Institute of Materials Science and Engineering (IMSE) at Washington University in St. Louis for providing access to electron microscopy facilities. S.S. acknowledges support from the National Science Foundation (CBET-1900277) and the National Institutes of Health (R21CA236652, R21EB030171). J.A.P. acknowledges support from the National Institutes of Health (2R01 AI087682 and R01 304AI130454). S.C.M. acknowledges support from the National Institutes of Health (R56AI104732). S.S. and A.S. conceived the project and designed the experiments. A.S. optimized and performed FluoroDOT assays and data analysis. J.A.P. E.M. and A.S. designed and performed the experiments for Mtb infection on BMDCs and BMDMs. J.L. helped with plasmonic-fluor optimization for the assay. S.K. developed the image-processing algorithm for particle counting and cluster identification. M.B.M. helped with acquiring PBMCs and performing and analyzing ELISpot and FluoroSPOT assays. S.C.M. and H.J. designed alveolar macrophage experiments and provided cells for the experiment. R.G. helped with antibody conjugation to plasmonic-fluor. P.R. acquired SEM images. Z.W. helped with BMDC isolation and calculation of molar concentration of plasmonic-fluor. J.J.M. helped with development of plasmonic-fluors. C.P.-C. and J.D.E. isolated and provided CD4+ Th1 effector cells. J.A.P. and S.S. supervised the project. A.S. J.A.P. and S.S. wrote the paper. All authors reviewed and commented on the manuscript. The authors declare the following competing financial interest(s): J.L. J.J.M. and S.S. are inventors on pending patent related to plasmonic-fluor technology, and the technology has been licensed by the Office of Technology Management at Washington University in St. Louis to Auragent Bioscience, LLC. J.L. J.J.M. and S.S. are co-founders/shareholders of Auragent Bioscience, LLC. These potential conflicts of interest have been disclosed and are being managed by Washington University in St. Louis. A.S. is currently working with Auragent Bioscience.
Funding Information:
We thank Dr. Nathaniel Huebsch from the Department of Biomedical Engineering at Washington University in St. Louis for providing access to the epifluorescence microscope used in this study. The authors thank Nano Research Facility (NRF) and Institute of Materials Science and Engineering (IMSE) at Washington University in St. Louis for providing access to electron microscopy facilities. S.S. acknowledges support from the National Science Foundation ( CBET-1900277 ) and the National Institutes of Health ( R21CA236652 , R21EB030171 ). J.A.P. acknowledges support from the National Institutes of Health ( 2R01 AI087682 and R01 304AI130454 ). S.C.M. acknowledges support from the National Institutes of Health ( R56AI104732 ).
Publisher Copyright:
© 2022 The Author(s)
PY - 2022/8/22
Y1 - 2022/8/22
N2 - Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we introduce the “FluoroDOT assay,” which uses an ultrabright nanoparticle plasmonic-fluor that enables high-resolution imaging of protein secretion. We find that plasmonic-fluors are 16,000-fold brighter, with nearly 30-fold higher signal-to-noise compared with conventional fluorescence labels. We demonstrate high-resolution imaging of different secreted cytokines in the single-plexed and spectrally multiplexed FluoroDOT assay that revealed cellular heterogeneity in secretion of multiple proteins simultaneously. Using diverse biochemical stimuli, including Mycobacterium tuberculosis infection, and a variety of immune cells such as macrophages, dendritic cells (DCs), and DC-T cell co-culture, we demonstrate that the assay is versatile, facile, and widely adaptable for enhancing biological understanding of spatial and temporal dynamics of single-cell secretome.
AB - Secreted proteins mediate essential physiological processes. With conventional assays, it is challenging to map the spatial distribution of proteins secreted by single cells, to study cell-to-cell heterogeneity in secretion, or to detect proteins of low abundance or incipient secretion. Here, we introduce the “FluoroDOT assay,” which uses an ultrabright nanoparticle plasmonic-fluor that enables high-resolution imaging of protein secretion. We find that plasmonic-fluors are 16,000-fold brighter, with nearly 30-fold higher signal-to-noise compared with conventional fluorescence labels. We demonstrate high-resolution imaging of different secreted cytokines in the single-plexed and spectrally multiplexed FluoroDOT assay that revealed cellular heterogeneity in secretion of multiple proteins simultaneously. Using diverse biochemical stimuli, including Mycobacterium tuberculosis infection, and a variety of immune cells such as macrophages, dendritic cells (DCs), and DC-T cell co-culture, we demonstrate that the assay is versatile, facile, and widely adaptable for enhancing biological understanding of spatial and temporal dynamics of single-cell secretome.
KW - Interleukin-1 beta
KW - Tumor Necrosis Factor-alpha
KW - cytokine secretion
KW - fluorescence imaging
KW - plasmonic nanoparticles
KW - single-cell secretion
KW - tuberculosis
UR - http://www.scopus.com/inward/record.url?scp=85136658899&partnerID=8YFLogxK
U2 - 10.1016/j.crmeth.2022.100267
DO - 10.1016/j.crmeth.2022.100267
M3 - Article
C2 - 36046626
AN - SCOPUS:85136658899
SN - 2667-2375
VL - 2
JO - Cell Reports Methods
JF - Cell Reports Methods
IS - 8
M1 - 100267
ER -