A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions

Artinger, Marc; Matti, Christoph; Gerken, Oliver J.; Veldkamp, Christopher T.; Legler, Daniel F.

A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions

Dateien

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Datum

2021

Autor:innen

Artinger, Marc

Matti, Christoph

Gerken, Oliver J.

Veldkamp, Christopher T.

Legler, Daniel F.

Open Access-Veröffentlichung

Open Access Gold

Sammlungen

Biologie

Publikationstyp

Zeitschriftenartikel

Publikationsstatus

Published

Erschienen in

International Journal of Molecular Sciences (IJMS). MDPI. 2021, 22(8), 4158. ISSN 1661-6596. eISSN 1422-0067. Available under: doi: 10.3390/ijms22084158

Zusammenfassung

Chemokines guide leukocyte migration in different contexts, including homeostasis, immune surveillance and immunity. The chemokines CCL19 and CCL21 control lymphocyte and dendritic cell migration and homing to lymphoid organs. Thereby they orchestrate adaptive immunity in a chemokine receptor CCR7-dependent manner. Likewise, cancer cells that upregulate CCR7 expression are attracted by these chemokines and metastasize to lymphoid organs. In-depth investigation of CCR7 expression and chemokine-mediated signaling is pivotal to understand their role in health and disease. Appropriate fluorescent probes to track these events are increasingly in demand. Here, we present an approach to cost-effectively produce and fluorescently label CCL19 and CCL21 in a semi-automated process. We established a versatile protocol for the production of recombinant chemokines harboring a small C-terminal S6-tag for efficient and site-specific enzymatic labelling with an inorganic fluorescent dye of choice. We demonstrate that the fluorescently labeled chemokines CCL19-S6^Dy649P1 and CCL21-S6^Dy649P1 retain their full biological function as assessed by their abilities to mobilize intracellular calcium, to recruit β-arrestin to engaged receptors and to attract CCR7-expressing leukocytes. Moreover, we show that CCL19-S6^Dy649P1 serves as powerful reagent to monitor CCR7 internalization by time-lapse confocal video microscopy and to stain CCR7-positive primary human and mouse T cell sub-populations.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Schlagwörter

fluorescent chemokine; CCL19; CCL21; CCR7; cell migration; leukocytes; flow cytometry; chemokine production

Zitieren

ISO 690

ARTINGER, Marc, Christoph MATTI, Oliver J. GERKEN, Christopher T. VELDKAMP, Daniel F. LEGLER, 2021. A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions. In: International Journal of Molecular Sciences (IJMS). MDPI. 2021, 22(8), 4158. ISSN 1661-6596. eISSN 1422-0067. Available under: doi: 10.3390/ijms22084158

BibTex

@article{Artinger2021-04-16Versa-54163,
  year={2021},
  doi={10.3390/ijms22084158},
  title={A Versatile Toolkit for Semi-Automated Production of Fluorescent Chemokines to Study CCR7 Expression and Functions},
  number={8},
  volume={22},
  issn={1661-6596},
  journal={International Journal of Molecular Sciences (IJMS)},
  author={Artinger, Marc and Matti, Christoph and Gerken, Oliver J. and Veldkamp, Christopher T. and Legler, Daniel F.},
  note={Article Number: 4158}
}

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