The sensitivity of replication-competent viruses to coreceptor inhibitors was determined using TZM-bl or SupT1/CCR5 cells. For TZM-bl cells, the cells were infected with viruses at 37°C for 2 days in the presence of various concentrations of coreceptor inhibitors. Luciferase activities of the cells were measured using a luminometer (Lumat LB 9501/16; Berthold). The sensitivity of the virus to coreceptor inhibitors was expressed as the 50% effective concentration (EC50), which was the drug concentration that reduced infection levels by 50% compared with that in the infected, drug-free control of triplicate experiments. For SupT1/CCR5 cells, 5×103 cells in U-bottom 96-well microplates were infected with the same amount of virus (100 TCID50) in the presence of various AMD3100 concentrations, and then cultured for 6 days. The cytopathic effect was determined using an MTT assay as described previously [37] (link).
Lumat lb 9501 16
Manufactured by Berthold Technologies
Sourced in Germany
The Lumat LB 9501/16 is a highly sensitive luminometer designed for the quantitative analysis of light-emitting reactions. It is capable of measuring luminescence in a wide range of applications, including biochemical, cell-based, and molecular biology assays.
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3 protocols using lumat lb 9501 16
1
Sensitivity of Viruses to Coreceptor Inhibitors
2
Transient Transfection of U937 Macrophages
3
Mitochondrial Assays in SH-SY5Y Cells
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Cell-based mitochondrial activity assays were performed using SH-SY5Y cells in 96-well black plates with clear bottom as described previously in detail22 (link),47 (link). The assays cover the quantitative assays for cell viability (calcein), complex 1, methyl thiazyl tetrazolium-mitochondrial dehydrogenase activity (MTT), mitochondrial membrane potential (TMRE, Molecular Probe, Eugene, OR, USA), ATP contents, and CM-H2DCFDA- or MitoSox (Molecular Probe)-dependent reactive oxygen species (ROS). The fluorescence intensities at 550 nm/580 nm for TMRE, at 510 nm/580 nm for Mito Sox, or 494 nm/522 nm for DCF-DA were normalized by Hoechst intensity at 355 nm/480 nm (Spectramax Gemini EM, Molecular Devices, Sunnyvale, CA, USA). The Caspase-Glo3/7 assay system (Promega Co, Madison, WI, USA) was used for caspase detection according to the manufacturer’s instructions. Briefly, the drug-pretreated SH-SY5Y cells were exposed to 1 mM MPP+ for 24 h, and subsequently incubated with the Caspase-Glo reagent containing proluminescent DEVD tetrapeptide substrate for 1 h. The luminescent intensity was measured by a luminometer (Lumat LB 9501/16, Berthold, Wildbad, Germany).
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