TY - JOUR
T1 - Labeling and tracking of immune cells in ex vivo human skin
AU - Dijkgraaf, Feline E.
AU - Toebes, Mireille
AU - Hoogenboezem, Mark
AU - Mertz, Marjolijn
AU - Vredevoogd, David W.
AU - Matos, Tiago R.
AU - Teunissen, Marcel B. M.
AU - Luiten, Rosalie M.
AU - Schumacher, Ton N.
N1 - Funding Information: Plasmid sequences for anti-mouse and anti-human CD8 nanobodies were kindly provided by 121Bio with support of M. Gostissa and G. Grotenbreg (Agenus subsequently acquired substantially all the assets of 121Bio). We acknowledge H. Ploegh (Harvard University) for the sortase expression vector and H. Ovaa and D. El Atmioui (Leiden University) for providing the GGGC peptide. We thank T. Venema (Slotervaart Ziekenhuis), P.G.L. Koolen (Rode Kruis Ziekenhuis), and W.G. van Selms (Onze Lieve Vrouwe Gasthuis West) and staff of the plastic surgery departments for the human skin tissue. We thank J. Beltman (LUMC) and B. van den Broek (Netherlands Cancer Institute, NKI) for analysis of migration parameters, J. Song (NKI) for histopathological analysis, M. Hoekstra (NKI) for illustration of the ex vivo imaging setup, T. Rademakers (Maastricht University), M. Rashidian (Dana-Farber Cancer Institute), L. Oomen, and L. Brocks (NKI) for technical support, and L. Perie (Curie Institute) and members of the Schumacher and Haanen laboratories for discussions. This work was supported by ERC AdG Life-His-T (to T.N.S.) and an EADV Research Fellowship (to T.R.M.). Publisher Copyright: © 2020, The Author(s), under exclusive licence to Springer Nature Limited.
PY - 2021/2
Y1 - 2021/2
N2 - Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8+ T cells, other immune cells of interest, or extracellular tissue components. Subsequent longitudinal imaging allows one to describe the dynamic behavior of human skin-resident cells in situ. In addition, this strategy can be used to study immune cell function in murine skin. The ability to follow the spatiotemporal behavior of CD8+ T cells and other immune cells in skin, including their response to immune stimuli, provides a platform to investigate physiological immune cell behavior and immune cell behavior in skin diseases. The mounting, staining and imaging of skin samples requires ~1.5 d, and subsequent tracking analysis requires a minimum of 1 d. The optional production of fluorescently labeled nanobodies takes ~5 d.
AB - Human skin harbors various immune cells that are crucial for the control of injury and infection. However, the current understanding of immune cell function within viable human skin tissue is limited. We developed an ex vivo imaging approach in which fresh skin biopsies are mounted and then labeled with nanobodies or antibodies against cell surface markers on tissue-resident memory CD8+ T cells, other immune cells of interest, or extracellular tissue components. Subsequent longitudinal imaging allows one to describe the dynamic behavior of human skin-resident cells in situ. In addition, this strategy can be used to study immune cell function in murine skin. The ability to follow the spatiotemporal behavior of CD8+ T cells and other immune cells in skin, including their response to immune stimuli, provides a platform to investigate physiological immune cell behavior and immune cell behavior in skin diseases. The mounting, staining and imaging of skin samples requires ~1.5 d, and subsequent tracking analysis requires a minimum of 1 d. The optional production of fluorescently labeled nanobodies takes ~5 d.
UR - http://www.scopus.com/inward/record.url?scp=85097879840&partnerID=8YFLogxK
U2 - https://doi.org/10.1038/s41596-020-00435-8
DO - https://doi.org/10.1038/s41596-020-00435-8
M3 - Article
C2 - 33349704
SN - 1754-2189
VL - 16
SP - 791
EP - 811
JO - Nature protocols
JF - Nature protocols
IS - 2
ER -