By Derek Kinchington, Raymond F. Schinazi
Royal London university of medication, united kingdom. moment writer, Raymond F. Schinazi, is at Emory Univ., Decatur, GA. deals simple and medical researchers types to guage compounds powerful opposed to acute and persistent infections. contains entire assays, caliber trying out, and unpublished equipment. DNLM: Hepatitis, Viral, Human - drug treatment.
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For the background count, 5 mL of scintillation solution is pipetted into an empty vial. 17a. For the total count, 5 mL of thoroughly mixed (inverted five times) separation suspension is pipetted into a 7-mL plastic screw-cap scintillation vial, centrifuged for 1 min at 500g, and the supernatant tipped off without losing the pellet. Then 50 µL of probe solution is added. b. Add 5 mL of scintillation solution to the vial and resuspend the pellet by vortexing. 18. All the vials are placed in the dark for at least 5 min, removed, and wiped with a clean, damp, paper towel and placed into a scintillation counter.
On d 3, wash the cells once with warmed DPBS and add fresh medium containing the appropriate compound to the wells. 5. After 24 h, transfer the supernatants to v-bottomed 96-well plates and remove cellular debris by centrifugation (15 min, 2500 rpm at 4°C, Sorvall RT-6000D). Transfer 90 µL of the clarified supernatants to new v-bottomed plates and store at –70°C for quantification of HBV DNA. 2. Cytotoxicity Assay 1. 1. 2. Incubate at 37°C for at least 1 h or until color develops. 3. Read the absorbance at 490 nm on a microplate reader to assess cell viability.
Cell nuclei will fluoresce. In mycoplasma-negative cultures, the nuclei will be seen against a dark background. In mycoplasma-positive cultures, the cell nuclei will be seen among fluorescing thread-like or coccal structures (Fig. 1). In an alternative system, cells of the test culture can be inoculated onto coverslips preinoculated with an indicator cell line, such as the Vero African Green Monkey cell line. In this case the Vero cell should be inoculated at a cell concentration of 1 × 104 cells/mL and left for 4–24 h prior to addition of the test sample.