The lymphatic ring assay: a 3D-culture model of lymphangiogenesis.

in Nature Protocols (2008)

Lymphangiogenesis, the formation of new lymphatic vessels, is associated to numerous pathologies1 and understanding the molecular and cellular basis of this complex process is essential for the ... [more ▼]

Lymphangiogenesis, the formation of new lymphatic vessels, is associated to numerous pathologies1 and understanding the molecular and cellular basis of this complex process is essential for the development of novel therapeutic strategies. Studies on lymphangiogenesis have been hampered by difficulties in culturing lymphatic capillaries as three-dimensional (3D) structures in vitro that mimic the in vivo features of lymphatic vessels and lymphangiogenesis. The lymphatic ring assay described here phenocopies the different steps of lymphangiogenesis, including the spreading from a preexisting vessel, cell proliferation, migration and differentiation into capillaries. It consists on the adaptation of the aortic ring assay that has proved to be useful to investigate the molecular basis of angiogenesis2-4. The lymphatic ring model is an ideal assay for testing the activity of lymphangiogenic agonists or antagonists. The absence of inflammatory cells allows a simple interpretation of results and the determination of direct effects of compounds on lymphatic endothelial cell properties. Another advantage of the lymphatic ring assay is that cell outgrowing are primary cells which have not been modified by repeated passages or immortalization. This culture model bridges the gap between in vitro and in vivo studies and allows genetic analysis by using thoracic ducts from genetically modified mice. [less ▲]

Atomic force microscopy of supported lipid bilayers.

in Nature Protocols (2008), 3(10), 1654-9

Supported lipid bilayers (SLBs) are widely used in biophysical research to investigate the properties of biological membranes and offer exciting prospects in nanobiotechnology. Atomic force microscopy ... [more ▼]

Supported lipid bilayers (SLBs) are widely used in biophysical research to investigate the properties of biological membranes and offer exciting prospects in nanobiotechnology. Atomic force microscopy (AFM) has become a well-established technique for imaging SLBs at nanometer resolution. A unique feature of AFM is its ability to monitor dynamic processes, such as the interaction of bilayers with proteins and drugs. Here, we present protocols for preparing dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC) bilayers supported on mica using small unilamellar vesicles and for imaging their nanoscale interaction with the antibiotic azithromycin using AFM. The entire protocol can be completed in 10 h. [less ▲]