Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy
| Autor: | William Fumey, Axel Leingärtner, Martin Trepel, Joanna Schmid, Peter Bannas, Björn Rissiek, Valentin Kunick, Alexander Lenz, Friedrich Koch-Nolte, Friedrich Haag, Gerhard Adam |
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| Rok vydání: | 2015 |
| Předmět: |
Pathology
medicine.medical_specialty Fluorescence-lifetime imaging microscopy General Immunology and Microbiology medicine.diagnostic_test General Chemical Engineering General Neuroscience Biology General Biochemistry Genetics and Molecular Biology Flow cytometry Transplantation In vivo Biophysics Fluorescence microscope medicine Molecular imaging Preclinical imaging Ex vivo |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| DOI: | 10.3791/52462 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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