By Vojo Deretic (auth.), Vojo Deretic (eds.)
Autophagy and phagocytosis are exact but in part morphologically related approaches. In Autophagosome and Phagosome, authoritative scientists current easy-to-follow tools on autophagy, a swiftly becoming box with a necessity for criteria of evaluate, and phagocytosis, a comparatively mature box that could gain significantly from up-to-date tools, as a way to urged additional explorations in their similarities and transformations. The equipment on autophagy enable the reader to discover acceptable innovations to spot, video display, and quantify autophagic approaches, whereas the tools dedicated to phagocytosis offer researchers with a number of smooth options for in vitro and in vivo reviews of phagosomal organelles. Following the winning Methods in Molecular Biology™ sequence layout, chapters comprise step by step laboratory protocols, lists of helpful fabrics, and advice for troubleshooting and averting recognized pitfalls.
Comprehensive and forward-thinking, Autophagosome and Phagosome deals a helpful advisor to either mobile approaches whereas inciting researchers to discover the possibly very important connections among the two.
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Extra resources for Autophagosome and Phagosome
The role of this autophagosome-associated ER, if any, is currently unknown. 2. The cytoplasmic contents of autophagosomes include organelles, such as ER membranes and mitochondria (Fig. 4A,B). Ribosomes are frequently seen inside autophagosomes (Figs. 4A,B, 5B, and 6A,B) and serve as a good marker structure for the cytoplasmic contents. The diameter of autophagosome profiles varies between 300 and 400 nm and several micrometers. In many cultured cells the average diameter is about 600 nm. Autophagosomes are frequently observed in fusion profiles with endosomal or lysosomal vesicles (18).
The lattice is created using the computer program. If the microscope is using a film camera, point counting can be performed using a light box. The lattice is printed on an overhead foil which is then placed on top of the film sheet. A magnifying loop can be used to help counting. 18 Eskelinen of the vacuoles (point counting 1; Fig. 2C). This can be done directly from photographic negatives if a digital camera is not available in the microscope. The cell area can be measured using about 400× magnification, which allows the whole grid square to fit in one photograph.
5–1 mL of Earle’s balanced salt solution (EBSS, nutrient-free [NF] medium). Finally, cells are incubated at 37ºC in 5% CO2 atmosphere for a variable time frame, depending on the experimental setting (see Note 2). 3. ). 2. Inhibition of Autophagy The cascade of events leading to autophagy can be suppressed at various steps (36) (Fig. 1), which include: (1) the formation of autophagosomes; (2) the fusion between autophagosomes and lysosomes (3,13); and (3) the final, lysosomal degradation phase (15,37).