By Vojo Deretic (auth.), Vojo Deretic (eds.)
Autophagy and phagocytosis are detailed but partly morphologically comparable approaches. In Autophagosome and Phagosome, authoritative scientists current easy-to-follow tools on autophagy, a swiftly growing to be box with a necessity for criteria of evaluate, and phagocytosis, a comparatively mature box that can gain significantly from up to date tools, so one can recommended extra explorations in their similarities and alterations. The equipment on autophagy permit the reader to discover applicable concepts to spot, visual display unit, and quantify autophagic approaches, whereas the equipment dedicated to phagocytosis offer researchers with numerous glossy ideas for in vitro and in vivo stories of phagosomal organelles. Following the winning Methods in Molecular Biology™ sequence structure, chapters contain step by step laboratory protocols, lists of important fabrics, and tips for troubleshooting and averting identified pitfalls.
Comprehensive and forward-thinking, Autophagosome and Phagosome bargains a worthwhile consultant to either mobile strategies whereas inciting researchers to discover the doubtless very important connections among the two.
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36 Tasdemir et al. lamp-1 lamp-2 Lysosome-associated membrane protein 1 Lysosome-associated membrane protein 2 NM_002294 NM_005561 NM_002647 sense 5’-GGCUGAAACUACCAGUAAAdTdT-3’ antisense 5’-UUUACUGGUAGUUUCAGCCdTdT-3’ sense 5’-GGAGGCAAAUAUCCAGUUAdTdT-3’ antisense 5’-UAACUGGAUAUUUGCCUCCdTdT-3’ sense 5’-CGAGAAAUGCAACACGUUAdTdT-3’ antisense 5’-UAACGUGUUGCAUUUCUCGdTdT-3’ sense 5’-GGAAUCCAGUUGAAUACAAdTdT-3’ antisense 5’-UUGUAUUCAACUGGAUUCCdTdT-3’ sense 5’-GCUGUGCGGUCUUAUGCAUdTdT-3’ antisense 5’-AUGCAUAAGACCGCACAGCdTdT-3’ sense 5’-GCGGUCUUAUGCAUUGGAAdTdT-3’ antisense 5’-UUCCAAUGCAUAAGACCGCdTdT-3’ (12) (12) (82) Notes: siRNAs are provided either as 100 mM stock solutions (to be stored as such at −20°C), or in dehydrated form.
Then use the wet spatula to transfer the gelatin and cells to a Petri dish on ice, and cut the cell pellet to small cubes/sticks (maximum side length about 2 mm). 20 Eskelinen Before freezing, the cells need to be incubated in a cryoprotectant. 7 M sucrose–15% PVP) are mostly used. PVP gives plasticity to the block and thus helps cryosectioning. Incubate the cell cubes in sucrose-PVP at 4°C, with occasional mixing, for several hours to overnight, until the cubes sink to the bottom. Trim the cubes under a stereo microscope and mount on cryoultramicrotome holders at 4°C (in a cold room, or using an ice bath).
Cell Biol. 25, 1025–1040. 4. Lum, J. , Bauer, D. , et al. (2005) Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell 120, 237–248. 5. , et al. (2004) The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036. 6. Eskelinen, E. , and Saftig, P. (2003) At the acidic edge: emerging functions for lysosomal membrane proteins. Trends Cell Biol. 13, 137–145. 7. , et al. (2000) Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2 -deficient mice.
Autophagosome and Phagosome by Vojo Deretic (auth.), Vojo Deretic (eds.)