HCV RNA replication kinetics were determined by using the HCV JcR2A reporter construct. Briefly, cells were collected at 4, 24, 48 and 72 h post-electroporation and lysed in luciferase lysis buffer (1% Triton X-100, 10% glycerol, 25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, and 1 mM DTT) for 15 min at RT. Cell lysates were transferred to 96-well plates and coelenterazine-containing luciferase assay buffer (25 mM glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, and 15 mM K3PO4, pH 7.8) was injected. Renilla luciferase activities were measured using a Mithras LB 940 plate luminometer (Berthold Technologies, Freiburg, Germany). Obtained values were normalized to the 4 h value of each transfection to correct for transfection efficiency. To measure the transmission of HCV, culture supernatants were used to inoculate naïve Huh7.5 cells, and after 72 h, cells were lysed and subjected to luciferase assay. NanoLuciferase (Nluc) activity was measured using the Nano-Glo Luciferase Assay System (Promega) according to the instruction of the manufacturer with slight modifications. In brief, 50 μl of samples were mixed with 50 μl NLuc substrate (1:1000) in the assay buffer and NLuc activities were measured using a Mithras LB 940 plate luminometer (Berthold Technologies, Freiburg, Germany).
Mithras lb940 plate luminometer
Manufactured by Berthold Technologies
Sourced in Germany
The Mithras LB940 is a multi-mode microplate luminometer designed for highly sensitive detection of luminescent signals. It offers automated plate handling and supports a range of microplate formats. The device is capable of measuring luminescence, fluorescence, and absorbance, providing a versatile platform for various bioassays and cellular analyses.
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3 protocols using mithras lb940 plate luminometer
1
Quantifying HCV RNA Replication and Transmission
2
Cell Cytotoxicity Quantification Assays
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Cytotoxicity was measured by using two different assays as specified in the results section. First, we used the CellTiter-Glo Luminescent Assay kit (Promega), quantifying intracellular ATP content, according to the manufacturer’s instructions. In brief, cells were lysed by adding an amount of CellTiter-Glo Reagent equal to the cell culture medium volume. After gentle mixing and 10 min incubation at room temperature, luminescence was measured using a Mithras LB940 plate luminometer (Berthold Technologies). Second, cell cytotoxicity was measured by WST-1 assay (Roche). Cells were seeded into 96 well-plates in triplicates and cultured for duration indicated in the result section. Culture medium was removed and cells were incubated in 100 μl/well of WST-reagent at 37°C for 30 min. Cell viability was analyzed by measuring absorbance at 450 nm using a Tecan XFluor4 reader (Wiesbaden, Germany).
3
Luciferase-Based HCV Replication Assay
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HCV replicons or genomes encoding firefly or Renilla luciferase, respectively, were transfected into Huh7-derived cells and viral replication was measured by luciferase assay as reported earlier (Reiss et al., 2011 (link); Stoeck et al., 2017 ). In brief, cells were lysed in luciferase lysis buffer (25 mM glycylglycin, 15 mM MgSO4, 4 mM EGTA, 1% Triton X-100, 10% glycerol, pH 7.8) and frozen at −20°C. Luminescence was measured after adding the respective substrate (coelenterazine for Renilla luciferase and luciferin, glycyl-glycine, ATP and dithiothreitol for firefly luciferase) by using a Mithras LB940 plate luminometer (Berthold Technologies).
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