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Интеллектуальная Система Тематического Исследования НАукометрических данных |
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Culturing of Dorsal Root Ganglia Explants ex vivo: 3-Dimentional visualization of axon growth, branching and neural cell migrationтезисы доклада
- Авторы: Semina EV, Rubina KA, Sysoeva VYu, Tkachuk VA
- Сборник: Super resolution in different dimentions. Advanced Microscopy Meeting. Abstract book
- Тезисы
- Год издания: 2015
- Место издания: Lomonosov Moscow State University Moscow Russia Moscow
- Первая страница: 82
- Последняя страница: 82
- Аннотация: The protocol describes isolation, culturing and immunofluorescent (IF) staining of axons in 3-dimentional ex vivo culture of dorsal root ganglion (DRG) explants in Matrigel (MG). Unlike other methods, this approach can be used to evaluate axonal growth and neural cell migration in a 3D culture. The method also allows to assess the impact of different agents on the growth and branching of neurites as well as to test the effect of these substances on cell migration - basic processes, reflecting regeneration of nerve tissue. To evaluate the rate of neurites elongation we used darkfield light microscopy; cell migration, axonal direction and branching were assessed using IF labeling and confocal imaging. For experiments, mice were anesthetized and DRG were isolated under aseptic conditions using Olympus SZX16 microscope, placed in chambered coverglass (Lab-Tek), and covered with the drop of MG. The samples were cultured in sterile humidified incubator. Imaging was performed using light microscopy (Zeiss Axoivert 2000) at low magnification within 2 weeks. Image processing (neurites length, number of migrated cells) was obtained with MetaMorph software (Meta Imaging Series). For staining samples were fixed, permeabilized, treated for blocking of non-specific binding and subsequently incubated with 1st (NF200 for mature axon) and 2nd (AlexaFluor 594) antibodies, nuclei counterstained by DAPI. Images were acquired by confocal laser scanning microscopy (TCS SP5, Leica) equipped with a Plan-Apo x10, 1.40 NA objective and 543-Argon laser. We tested the impact of exogenously added urokinase (uPA at 10 ng/ml) on the directed growth and branching of axons, spontaneous cell migration and the rate cell migration from DRG to MG. uPA administration significantly increased both, neurite outgrowth and cell migration. Thus, uPA stimulates axonal elongation (1.5 times longer compared to control, p<0.05) and activates spontaneous cell migration (2 times compared to control, p<0.05).
- Добавил в систему: Семина Екатерина Владимировна