| Popis: |
Among the central constituents of the innate immune system are macrophages, which are known for phagocytosis or ‘eating’ foreign particles or pathogens. Macrophages express several cell-surface molecules including Toll-like receptors (TLR), and scavenger receptors which play a vital role in their response to pathologen stimuli. Studies suggest that macrophages exhibit age-related deficiencies in TLR function, and as a result elderly are more susceptible to infections. Previous investigations in aged populations have revealed an increase in the number of bone marrow macrophages that have an impaired ability to produce or release cytokines. The increase in macrophage numbers may reflect a compensation for their reduced function. Our hypothesis is that the reduced function of the receptors is due to the effect of aging on fluidity and rigidity of the membrane. Therefore, we aim to measure the mobility of macrophage receptors in young and old individuals to investigate whether there are significant differences in membrane fluidity, and if that impacts receptor function. Our experimental methodology includes imaging fluorescently labeled membrane receptors on macrophages isolated from mice peritoneum to track their displacement. Single-particle tracking has been successfully utilized in previous studies to track the trajectories of molecules in the cell membrane, and total internal reflection fluorescence microscopy (TIRF) has enabled visualization of single molecules inside living cells. In this study, we have captured a sequence of images for each cell using TIRF, and have analyzed the images with a custom routine algorithm to detect and localize the receptors to calculate their diffusion coefficient using mean square displacement (MSD) as the most common measure of motion. To our knowledge, this is the first study that has used this technique to compare receptors’ diffusion through the membrane of macrophages for young and old organisms. |
