title: Validation of nanobody and antibody based in vivo tumor xenograft NIRF-imaging experiments in mice using ex vivo flow cytometry and microscopy.
abstract: This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies. First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results. The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.
authors: Department of Diagnostic and Interventional Radiology, University Medical Center, Hamburg; p.bannas@uke.de.
authors: Peter
authors: P
authors: Bannas
authors: Department of Diagnostic and Interventional Radiology, University Medical Center, Hamburg; Institute of Immunology, University Medical Center, Hamburg.
authors: Alexander
authors: A
authors: Lenz
authors: Department of Diagnostic and Interventional Radiology, University Medical Center, Hamburg; Institute of Immunology, University Medical Center, Hamburg.
authors: Valentin
authors: V
authors: Kunick
authors: Department of Diagnostic and Interventional Radiology, University Medical Center, Hamburg; Institute of Immunology, University Medical Center, Hamburg.
authors: William
authors: W
authors: Fumey
authors: Institute of Immunology, University Medical Center, Hamburg.
authors: Björn
authors: B
authors: Rissiek
authors: Department of Diagnostic and Interventional Radiology, University Medical Center, Hamburg; Institute of Immunology, University Medical Center, Hamburg.
authors: Joanna
authors: J
authors: Schmid
authors: Institute of Immunology, University Medical Center, Hamburg.
authors: Friedrich
authors: F
authors: Haag
authors: University Cancer Center Hamburg, University Medical Center, Hamburg.
authors: Axel
authors: A
authors: Leingärtner
authors: Department of Oncology and Hematology, University Medical Center, Hamburg.
authors: Martin
authors: M
authors: Trepel
authors: Department of Diagnostic and Interventional Radiology, University Medical Center, Hamburg.
authors: Gerhard
authors: G
authors: Adam
authors: Institute of Immunology, University Medical Center, Hamburg.
authors: Friedrich
authors: F
authors: Koch-Nolte
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000032:analysis / Q000737:chemistry*; D064593:Heterografts / Q000473:pathology*; D006801:Humans; D008223:Lymphoma / Q000473:pathology*; D051379:Mice; D008819:Mice, Nude; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D057054:Molecular Imaging / Q000379:methods; D009368:Neoplasm Transplantation; D015203:Reproducibility of Results; D061905:Single-Domain Antibodies / Q000032:analysis / Q000737:chemistry*; D014183:Transplantation, Heterologous
chemical_list: D000906:Antibodies; D005456:Fluorescent Dyes; D061905:Single-Domain Antibodies