Antiviral Methods and Protocols by Derek Kinchington, Raymond F. Schinazi

By Derek Kinchington, Raymond F. Schinazi

Royal London institution of drugs, united kingdom. moment writer, Raymond F. Schinazi, is at Emory Univ., Decatur, GA. bargains easy and medical researchers types to guage compounds powerful opposed to acute and protracted infections. comprises entire assays, caliber checking out, and unpublished tools. DNLM: Hepatitis, Viral, Human - drug remedy.

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1. “Primary” or “Screening” Assay (96-Well Plate Format) This assay format is well suited to the screening of test compounds for potential antiviral activity. ). , 3TC). After treating for 9 d, the media are harvested from the antiviral plates and transferred to 96-well U-bottomed plates. They are then centrifuged, and supernatant is transferred to tubes for dot-blot hybridization analysis of HBV virion DNA. The medium is aspirated off of the toxicity plates and discarded. Toxicity plates are then incubated with neutral red dye (methylthiouracil [MTT] can also be used if preferred), washed with DPBS, developed with an acetic acid/ ethanol solution, and assayed in a plate reader.

4 (molecular biology grade). Chloroform. HBV Assays for Antiviral Activity 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 53 Isopropanol. Ammonium acetate. 25% bromophenol blue, 30% glycerol in TE. TAE (Tris-acetate-EDTA running buffer). Agarose (standard low–mr). Plastic pipets (1 mL to 50 mL, sterile, individually wrapped). 5 in. Glass Pasteur pipets. 96-Well tissue culture plates. 24-Well tissue culture plates. Tissue culture flasks (75, 150 cm2).

Of colonies counted × 100)/(dilution factor) (1) 5. Elimination of Contamination In the event of cultures becoming infected with mycoplasma, the best course of action is to discard the cultures and following extensive decontamination of the tissue culture cabinets and work surfaces, thereby resuscitating “clean” cell stocks. However, in the case of irreplaceable stocks this may not be practical. 1. Elimination of Contamination 1. Culture cells in the presence of the chosen antibiotic(s) for a period of 10–14 d, during which time most cultures will be passaged approx 4 times.

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