By Haitao Guo, Andrea Cuconati
This quantity serves as a reference for the dissemination of advances made within the examine of Hepatitis B Virus (HBV). Hepatitis B Virus: tools and Protocols info protocols and methods starting from cellphone tradition reports to in vivo and scientific immunology. The chapters during this publication talk about remedies of in vitro an infection structures, research and quantification of cccDNA and its mutations; in vitro polymerase task assays; mobile trafficking of middle proteins; intracellular calcium metabolism; detection, cloning, and sequencing of HBV markers; and new ideas aimed toward exploiting new mechanisms for drug discovery. The publication additionally covers classical equipment for answer of extracellular viral debris by way of local gel electrophoresis, and techniques for detecting HBV antigens in drug discovery. Written within the hugely profitable Methods in Molecular Biology series structure, chapters contain introductions to their respective subject matters, lists of the required fabrics and reagents, step by step, without problems reproducible laboratory protocols, and tips about troubleshooting and warding off recognized pitfalls.
Cutting-edge and entire, Hepatitis B Virus: equipment and Protocols is a helpful device for researchers to exploit towards their complex reports in HBV.
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2 Nucleus Staining 1. Wash the cells twice with 400 μL wash buffer. 2. Dilute Hoechst 33342 in DMEM (10 μg/mL) and add 200 μL to each well. 3. Incubate cells for 15 min at 37 °C and 95 % humidity. 4. Wash cells three times with ice cold PBS++. 2, prepare ice cold DMEM supplemented with 10–200 nM Myr-preS1-AX594. Precool transfected and non-transfected cells for 15 min by incubating with ice cold DMEM in a fridge at 4 °C. Discard supernatant and incubate transfected and non- transfected cells with 100 μL ice cold peptide solution for 30 min in a fridge at 4 °C (see Note 1).
J Hepatol 36(6):832–850 5. Mukhopadhayay S, Ananthanarayanan M, Stieger B, Meier PJ, Suchy FJ, Anwer MS (1997) cAMP increases liver Na + -taurocholate cotransport by translocating transporter to plasma membranes. Am J Physiol 273(4 Pt 1):G842–G848 6. Mühlfeld S, Domanova O, Berlage T, Stross C, Helmer A, Keitel V, Häussinger D, Kubitz R (2012) Short-term feedback regulation of bile salt uptake by bile salts in rodent liver. Stross C, Kluge S, Weissenberger K, Winands E, Häussinger D, Kubitz R (2013) A dileucine motif is involved in plasma membrane expression and endocytosis of rat sodium taurocholate cotransporting polypeptide (Ntcp).
Typically, at least 100 MGE (multiplicity of genome equivalents) is required for an efficient HBV infection. Here, we describe the infection of HepaRG, HepaRG-hNTCP, or PHH cells in a 24-well plate. 1. For each well, mix 50 μl 40 % PEG with 450 μl Differentiation medium. The final PEG concentration is 4 % (see Note 20). Vortex for 5 s. 2. Add desired amount of HBV, and vortex again for 5 s. Usually, 20 μl concentrated virus from HepAD38 can result in a well- detectable infection. Aspirate cell culture medium, then add inoculum, and refer as day 0.