RESUMO
The eukaryotic cell cycle is comprised of different phases that take place sequentially once, and normally only once, every division cycle. Such a dynamic process is best viewed in real time in living dividing cells. The insights that can be gained from such methods are considerably larger than any alternative technique that only generates snapshots. A great number of studies can gain from live cell imaging; however this method often feels somewhat intimidating to the novice. The purpose of this chapter is to demonstrate that imaging cell cycle phases in living cells from yeast to human is relatively easy and can be performed with equipment that is available in most research institutes. We present the different approaches, review different types of reporters, and discuss in depth all the aspects to be considered to obtain optimal results. We also describe our latest cell cycle markers, which afford unprecedented "sub"-phase temporal resolution.
Assuntos
Ciclo Celular , Imagem Molecular/métodos , Saccharomycetales/citologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Humanos , Camundongos , Células NIH 3T3RESUMO
Excessive bone resorption is frequently associated with chronic infections and inflammatory diseases. Whereas T cells were demonstrated to facilitate osteoclastogenesis in such diseases, the role of dendritic cells, the most potent activators of naive T cells, remains unclear. Using a model involving inflammation-driven alveolar bone loss attributable to infection, we showed that in vivo ablation of Langerhans cells (LCs) resulted in enhanced bone loss. An increased infiltration of B and T lymphocytes into the tissue surrounding the bone was observed in LC-ablated mice, including receptor activator of NF-κB ligand (RANKL)-expressing CD4(+) T cells with known capabilities of altering bone homeostasis. In addition, the absence of LCs significantly reduced the numbers of CD4(+)Foxp3(+) T-regulatory cells in the tissue. Further investigation revealed that LCs were not directly involved in presenting antigens to T cells. Nevertheless, despite their low numbers in the tissue, the absence of LCs resulted in an elevated activation of CD4(+) but not CD8(+) T cells. This activation involved elevated production of IFN-γ but not IL-17 or IL-10 cytokines. Our data, thus, reveal a protective immunoregulatory role for LCs in inflammation-induced alveolar bone resorption, by inhibiting IFN-γ secretion and excessive activation of RANKL(+)CD4(+) T cells with a capability of promoting osteoclastogenesis.