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The research field “osteoimmunology” comprises of two main scientific areas: the musculoskeletal system and immunology. Thus, for the investigation of our research questions, we are applying laboratory methods from both scientific fields. In the following, some commonly used methods are depicted:
X-ray imaging techniques as well as µCT analyses (Bruker Skyscan, Scanco VivaCT) were used in order to obtain an in-depth look into the structure and constitution of bone. Dependent on the research question, a resolution of 1 – 10µm can be applied for the analyses.
Evaluation of the immune cell composition of different tissues is another main interest in our research. Using flow cytometry, characteristics of different types of immune cells can be analyzed by using up to 18 different fluorescence colors (LSRFortessa).
We are applying different techniques in order to isolate primary cells from tissue samples (peripheral blood, spleen, lymph notes or bone marrow) in the cell culture: Isolation by using magnetic beads (MACS, magnetic activated cell sorting) or by using cell strainer (PluriSelect). Subsequently, isolated cells are directly analyzed or cultivated in vitro and further stimulated or differentiated.
Using different assay types, we are focusing on the following functions of the isolated cells in our lab: migration, angiogenesis, osteogenesis, adipogenesis, myogenesis and activity.
Expression analyses enable a deeper look into the activity and function of cells. Active transcribed genes can be detected by messenger RNA. This messenger RNA is isolated and transcribed into copy DNA (cDNA). Using specific designed primer, “targets (genes) of interest” are duplicated and further quantitatively evaluated. Expression analyses are performed by using Real Time quantitative PCR (RT qPCR) or the fluidigm card system.
We are applying specific antibodies in order to detect and also quantify the expression of proteins of interest, which are present on/in the analyzed cells at a certain time point. For these investigations, the ELISA system (enzyme linked immunesorbent assay) is used, either as a multi-array or sandwich approach.
Tissue sections for histological evaluation are produced by using the cryo or paraffin embedding technique. The following staining methods are applied to answer our research questions:
- Overview stainings like Movat Pentachrome, Sirius Red, McNeal, H&E, Giemsa or van Kossa are used to get an impression of the overall tissue structure and composition. For further knowledge, these stainings are investigated by using specific macroinstructions and thus a computer-assisted evaluation.
- Immunohistochemical stainings are used to analyze the appearance and distribution of specific cell subsets of the immune and bone systems, like T cells, B cells, macrophages, osteoclasts and osteoblasts. Furthermore, also vessels, cytokines as well as fibers of the connective tissue are of interest. For these analyses, predominantly fluorescence coupled antibodies are applied and the evaluation is performed at a confocal microscope (LSM 710).