By Vojo Deretic (auth.), Vojo Deretic (eds.)
Autophagy and phagocytosis are detailed but in part morphologically related procedures. In Autophagosome and Phagosome, authoritative scientists current easy-to-follow equipment on autophagy, a swiftly becoming box with a necessity for criteria of overview, and phagocytosis, a comparatively mature box that may gain significantly from up to date equipment, so one can steered extra explorations in their similarities and adjustments. The equipment on autophagy let the reader to discover acceptable ideas to spot, video display, and quantify autophagic approaches, whereas the tools dedicated to phagocytosis supply researchers with a number of sleek recommendations for in vitro and in vivo reports of phagosomal organelles. Following the profitable Methods in Molecular Biology™ sequence structure, chapters contain step by step laboratory protocols, lists of valuable fabrics, and suggestions for troubleshooting and heading off identified pitfalls.
Comprehensive and forward-thinking, Autophagosome and Phagosome bargains a priceless advisor to either mobile tactics whereas inciting researchers to discover the possibly very important connections among the two.
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Extra resources for Autophagosome and Phagosome
Principles of autophagic vacuole quantitation and point counting. (A) Grid squares can be used as sampling units. It is recommendable to count at least three squares from each sample. The selected squares should be uniformly distributed over the section. When a small magnification (400–600×) is used for this selection, selection bias is easier to avoid because autophagic vacuoles are not visible at low magnification. Fine Structure of the Autophagosome 17 9. Transfer the pellets to fresh resin in embedding molds and incubate for 4–6 h, RT.
Epon® epoxy resin (Miller-Stephenson, Danbury, CT). 14. Film processor: Curix 60 table-top film processor (Agfa, Mortsel, Belgium). 15. 19% picric acid (Sigma-Aldrich) in PBS. 5 mL of PBS, let cool to room temperature (RT), add 38 μL of saturated picric acid (Sigma-Aldrich), and move the fixative solution to ice bath (see also Notes 13–15). 16. Fluorescence microscope: IRE2 microscope equipped with a DC300F camera (Leica Microsystems GmbH, Wetzlar, Germany). 17. Fluorescent dyes used for the detection of autophagy are listed in Table 5.
With H2 O2 ) (35). 1. Wild-type HeLa cells are cultured in appropriate growth medium and passaged when approaching to confluence with trypsin/EDTA to provide new maintenance cultures in 175-cm2 flasks and experimental cultures. The latter are performed 44 Tasdemir et al. signal Inducers Endogenous modulators Plasma membrane signal Phagophore Cytoplasmic organelles Atg5 Atg6/Beclin-1 Atg10 Atg12 LAMP-2 hVps34 Inhibitors Endogenous modulators Bcl-2 Class I PI3K mTOR Chemical inhibitors Chemical inducers Brefeldin-A Carbamazepine Ceramide L-690,330 Lithium chloride Rapamycin Thapsigargin Tunicamycin Xestospongin B Xestospongin C Lysosome 3-methyladenine Bafilomycin A1 Hydroxychloroquine AV1 fusion Adaptive response to AV2 siRNAs Atg5 Atg6/Beclin-1 Atg10 Atg12 LAMP-2 hVps34 Bacterial infection Irradiation Oxidative stress Protein aggregates Starvation Viral infection degradation Fig.
Autophagosome and Phagosome by Vojo Deretic (auth.), Vojo Deretic (eds.)