By Vojo Deretic (auth.), Vojo Deretic (eds.)
Autophagy and phagocytosis are unique but partly morphologically related procedures. In Autophagosome and Phagosome, authoritative scientists current easy-to-follow tools on autophagy, a speedily becoming box with a necessity for criteria of evaluate, and phagocytosis, a comparatively mature box that may gain drastically from up to date tools, for you to recommended additional explorations in their similarities and changes. The tools on autophagy let the reader to discover acceptable options to spot, video display, and quantify autophagic approaches, whereas the equipment dedicated to phagocytosis offer researchers with a number of sleek concepts for in vitro and in vivo reports of phagosomal organelles. Following the profitable Methods in Molecular Biology™ sequence layout, chapters contain step by step laboratory protocols, lists of beneficial fabrics, and assistance for troubleshooting and fending off identified pitfalls.
Comprehensive and forward-thinking, Autophagosome and Phagosome bargains a necessary consultant to either mobile procedures whereas inciting researchers to discover the doubtless very important connections among the two.
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4. 5-mL, 12 × 75 mm FACS tubes (BD Falcon). 5. 6-, 12-, 24-well plates for cell culture (Corning Inc. Life Sciences). 6. 76 × 26 mm slides for fluorescence microscopy (Carl Roth GmbH, Karlsruhe, Germany). 2. Solutions 1. 5 g/L glucose, 4 mM l-glutamine and 110 mg/L sodium pyruvate (Gibco-Invitrogen, Carlsbad, CA) supplemented with 100 mM HEPES buffer (Gibco-Invitrogen) and 10% fetal bovine serum (FBS, from PAA Laboratories GmbH, Pasching, Austria). 2. 4 with 2 N NaOH. 3. 38 g/L (1mM) EDTA•4Na in Hank’s balanced salt solution (HBSS) (Gibco-Invitrogen).
G. 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.
To promote the From: Methods in Molecular Biology, vol. 445: Autophagosome and Phagosome Edited by: V. Deretic © Humana Press, Totowa, NJ 29 30 Tasdemir et al. degradation of their luminal content, autophagosomes fuse with lysosomes, thus forming the so-called autophagolysosomes (2). , due to a decreased fusion of AV with lysosomes) suffices to increase their number severalfold (3). Accordingly, ultrastructural studies have to be accompanied by evidence for increased protein and/or organelle turnover to ascertain increased autophagic activity.