By Vojo Deretic (auth.), Vojo Deretic (eds.)
Autophagy and phagocytosis are specified but in part morphologically related methods. In Autophagosome and Phagosome, authoritative scientists current easy-to-follow equipment on autophagy, a swiftly transforming into box with a necessity for criteria of overview, and phagocytosis, a comparatively mature box which may gain enormously from up-to-date equipment, with a view to recommended additional explorations in their similarities and adjustments. The tools on autophagy permit the reader to discover applicable thoughts to spot, computer screen, and quantify autophagic procedures, whereas the equipment dedicated to phagocytosis offer researchers with numerous sleek ideas for in vitro and in vivo experiences of phagosomal organelles. Following the profitable Methods in Molecular Biology™ sequence structure, chapters comprise step by step laboratory protocols, lists of beneficial fabrics, and information for troubleshooting and fending off recognized pitfalls.
Comprehensive and forward-thinking, Autophagosome and Phagosome deals a priceless advisor to either mobile methods whereas inciting researchers to discover the possibly vital connections among the two.
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Thereafter, cells are cultured at 37ºC in a 5% CO2 incubator. 3. ). 2. siRNA-Mediated Inhibition of Autophagy 1. HeLa cells are seeded in 6-well plates (approximately, 200 103 cells in 3 mL of growth medium), in order to obtain confluence levels around 60–70% on the next day. Methods for Assessing Autophagy 47 2. If cells have reached appropriate confluence, transfection of siRNA is performed 12–24 h after plating (see Note 3). For the delivery of siRNAs to adherent cells, liposome-based transfection is employed.
The cells should be semi-confluent. 4 at RT for 2 h. Scrape the cells from culture plate using a razor blade after 30 min fixation. Pellet the cells at full speed in an Eppendorf centrifuge for 5 min. Continue fixation as a pellet. Do not resuspend the pellet during the rest of embedding, but use gentle mixing during incubations. 2. 2 M HEPES (and store at 4°C). 3. Wash in PBS three times. 4. Postfix the pellets in 1% osmium tetroxide in water at RT for 1 h. 5. Wash in water twice. 6. Stain the pellets in 2% uranyl acetate in water at RT, in the dark, for 1 h.
22. 1 g of lead(II) citrate tribasic trihydrate (Sigma-Aldrich) in 100 mL dH2 O; add dropwise 10 N NaOH (Sigma-Aldrich) until solution becomes clear; store at RT. 23. Lipofectamine™ 2000 transfection reagent (Lipofectamine™, from GibcoInvitrogen). 24. 05% (w/v) bromophenol blue in dH2 O (or commercially available from Fermentas, Ontario, Canada). 25. l-Valine (Sigma-Aldrich), stock solution in dH2 O, at the concentration of 100 mM (stored at 4°C). 26. Lysis buffer: CelLytic™ M (Sigma-Aldrich). 27.
Autophagosome and Phagosome by Vojo Deretic (auth.), Vojo Deretic (eds.)