By Haitao Guo, Andrea Cuconati
This quantity serves as a reference for the dissemination of advances made within the learn of Hepatitis B Virus (HBV). Hepatitis B Virus: equipment and Protocols information protocols and methods starting from telephone tradition stories to in vivo and scientific immunology. The chapters during this booklet speak about remedies of in vitro an infection platforms, research and quantification of cccDNA and its mutations; in vitro polymerase job assays; mobile trafficking of center proteins; intracellular calcium metabolism; detection, cloning, and sequencing of HBV markers; and new recommendations aimed toward exploiting new mechanisms for drug discovery. The e-book additionally covers classical equipment for solution of extracellular viral debris by way of local gel electrophoresis, and strategies for detecting HBV antigens in drug discovery. Written within the hugely profitable Methods in Molecular Biology series structure, chapters contain introductions to their respective themes, lists of the mandatory fabrics and reagents, step by step, effortlessly reproducible laboratory protocols, and pointers on troubleshooting and keeping off recognized pitfalls.
Cutting-edge and accomplished, Hepatitis B Virus: equipment and Protocols is a worthwhile software for researchers to exploit towards their complicated reviews in HBV.
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Extra resources for Hepatitis B Virus: Methods and Protocols
1). 2. 2) and untransfected HepG2 (as control), add 150 μL of cell suspension to each Ibidi-well, and distribute the cells by gentle shaking. After overnight culturing at 37 °C and 95 % humidity 70–80 % final density should be reached (see Note 2). 1 Preliminaries 1. 7, respectively. Precool wash buffers and DMEM in 50 mL Falcon tubes on ice. 3. Prewarm DMEM to 37 °C in 50 mL tube. Prewarm microscope stage to 37 °C by switching on the air stream heater several hours before imaging to avoid focus shift.
1. Seed 3 × 106 HuH7 cells (see Note 11) in 10-cm dish with Culture medium. On the next day, transfect proximally 70 % confluent HuH7 cells (see Note 12) with transfection reagents such as lipofectamine 2000 or JetPrime according to the manufacturer’s protocol and refer as day 0. 3. Refresh the medium at days 3 and 8. Collect culture supernatant between day 3–8 and day 8–12. Centrifuge the collected supernatants at 3000 × g for 15 min at 4 °C to remove cell debris. Add 40 % PEG to a final concentration of 6 % (w/v) and prepared virus as mentioned above for HepAD38 cells.
4. Refresh the Differentiation medium every 2–3 days (see Note 15). 5. The HepaRG cells are ready for infection at day 28 (see Note 16). 2 Plating of PHHs for HBV Infection PHHs can be prepared from the liver tissue if the respective infrastructure and technique are available on site. , Biopredic) providing plated or cryopreserved platable human hepatocytes. The cell viability should be above 75 % before seeding. The following protocol describes the seeding of PHHs in a 24-well plate. 1. Dilute collagen in PBS to a final concentration of 100 μg/ml.