For the luciferase reporter assay, BPH-1 cells were plated in 48-well plates 24 h before transfection; then, the cells were transfected with the luciferase reporter using the TranslT-LT1 transfection reagent (Mirus Bio, Madison, WI, USA) according to the manufacturer’s instructions. For reporter quantitation, cell lysates were assayed using the Luciferase Assay System (Promega, Madison, WI, USA) and a TriStar LB941 Luminometer (Berthold technologies, Bad Wildbad, Germany) according to the manufacturer’s instructions.
Tristar lb 941 luminometer
Manufactured by Berthold Technologies
Sourced in Germany, United States, France
The Tristar LB 941 luminometer is a compact, high-performance instrument designed for the measurement of luminescence-based assays. It features a photomultiplier tube detector that enables sensitive and accurate detection of light signals. The Tristar LB 941 is capable of performing various luminescence-based analyses, including but not limited to bioluminescence, chemiluminescence, and fluorescence measurements.
Automatically generated - may contain errors
Lab products found in correlation
Passive lysis buffer (plb), by Promega (15 mentions)
96 well luminometer compatible tissue culture plates, by PerkinElmer (10 mentions)
Renilla lysis buffer, by Promega (5 mentions)
Renilla luciferase assay system, by Promega (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (3 mentions)
Western lightning oxidizing and luminol reagents, by PerkinElmer (3 mentions)
Maxisorp, by Thermo Fisher Scientific (3 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Cytotox one homogeneous membrane integrity assay, by Promega (2 mentions)
Cell culture lysis reagent, by Promega (2 mentions)
Dual luciferase assay kit, by Promega (2 mentions)
Luciferase assay kit, by Promega (2 mentions)
Celltiter glo luminescent cell viability assay, by Promega (2 mentions)
D luciferin potassium salt, by Thermo Fisher Scientific (2 mentions)
Celltiter glo one solution assay, by Promega (2 mentions)
Hrp conjugated anti igg secondary abs, by Thermo Fisher Scientific (2 mentions)
Western lightning ecl reagent, by PerkinElmer (2 mentions)
Maxisorp nunc plates, by Thermo Fisher Scientific (2 mentions)
Translt lt1 transfection reagent, by Mirus Bio (1 mentions)
Luciferase assay system, by Promega (1 mentions)
48 protocols using tristar lb 941 luminometer
1
Luciferase Reporter Assay in BPH-1 Cells
2
Transfection and Reporter Assay in HEK293T Cells
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HEK293T cells were plated at a density of 5 × 105 cells in 2 mL per well in 6-well plates and after 22–24 h, the calcium phosphate method was used to transfect the cells with 200 ng of pCSK-LacZ (Condie et al., 1990 (link)), with or without 1500 ng of Igκ2-IFN-LUC (Pomerantz et al., 2002 (link)), with or without the indicated amount of the pcDNA3 myc-CARD11 constructs, and the empty pcDNA3 vector to achieve 3000 ng of total DNA per condition. At 24 h post transfection, the media was replaced with 2 mL fresh complete DMEM and at 40–44 h post transfection, the cells were harvested in 400 μL 1× Reporter Lysis Buffer followed by incubation on ice for 10 min and centrifugation at 13,000 rpm for 10 min at 4°C. The chemiluminescent β-gal reporter gene assay (Roche, 11758241001) was performed using 2 μL of lysate according to the manufacturer's instructions using a Berthold Technologies TriStarLB 941 luminometer, integrating for 10 s after a 2 s delay. The measured β-gal activity was used to normalize for transfection efficiency and extract recovery and the normalized lysates were resolved by SDS-PAGE, transferred to PVDF membranes, and analyzed by western blotting with the mouse anti-myc primary antibody (Santa Cruz, sc-40) and the sheep anti-mouse (Cytiva, NA931V) secondary antibody to determine relative expression. Western blots represent one of at least two replicate experiments.
3
HCoV-229E-Luc Infection Assay in Huh-7 Cells
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HCoV-229E-Luc was first mixed with the compounds at the appropriate concentrations. On the day before infection, 6 × 104 Huh-7 cells and Huh-7/TMPRSS2 cells were seeded in 96-well plates at 37 °C. The cells were inoculated with HCoV-229E-Luc at a MOI of 0.5 in a final volume of 50 μL for 1 h at 37 °C in the presence of the different compounds, either at 25 μg/mL for the screening experiment or at increasing concentrations for the dose–response experiment. The virus was removed and replaced with culture medium containing the different compounds for 6 h at 37 °C. The cells were lysed in 20 μL of Renilla Lysis Buffer (Promega, Madison, WI, USA), and luciferase activity was quantified using a Tristar LB 941 luminometer (Berthold Technologies, Bad Wildbad, Germany) with the Renilla Luciferase Assay System (Promega) as recommended by the manufacturer.
4
Antiviral Activity Determination Protocol
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The antiviral activity of the compounds was determined by pre-incubating 104 TZMbl cells/well in a 96-well plate for 2 h at 37 °C and 7 % CO2 to maintain the optimal pH with or without a serial dilution of compounds. Next, 200 TCID50 of HIV-1 BaL virus was added to each well, and cultures were incubated for 48 h before luciferase activity was quantified. To this end, 120 μL of supernatant was removed, 75 μl of the luciferase substrate Steadylite (Perkin Elmer, Life Sciences, Zaventem, Belgium) was added to the wells, and the plates were incubated at room temperature on an orbital shaker for 10 min. Next, the luciferase activity was measured using a TriStar LB941 luminometer (Berthold Technologies GmbH & Co. KG., Bad Wildbad, Germany) and expressed in relative light units (RLU).
Each compound was tested in triplicate and each experiment was repeated in three independent runs. Antiviral activity was expressed as the percentage of viral growth compared to the control and plotted against the compound concentration. Next, non-linear regression analysis was used to calculate the EC50, using GraphPad Prism 5.03 using non-linear regression (GraphPad Software, San Diego, CA, USA).
Each compound was tested in triplicate and each experiment was repeated in three independent runs. Antiviral activity was expressed as the percentage of viral growth compared to the control and plotted against the compound concentration. Next, non-linear regression analysis was used to calculate the EC50, using GraphPad Prism 5.03 using non-linear regression (GraphPad Software, San Diego, CA, USA).
5
Knockdown of LARP Proteins in HEK293 Cells
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HEK293 cells were transfected with siRNAs targeting LARP4B, LARP4 or LARP1 and combinations thereof. For control a siRNA against GFP was used. Forty-eight hours post-transfection a pHA-FF reporter plasmid was transfected. Five hours later cell extracts were prepared as described above and luciferase activity was determined by using a standard chemoluminescence detection procedure at a TriStar LB941 luminometer (Berthold Technologies).
6
Measuring Viral Polymerase Activity
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To measure viral polymerase activity, 6 × 105 HEK293T cells were seeded in 6-well plates and co-transfected with 0.5 µg pHW2000 vector constructs encoding PB2, PB1, PA and NP genes of SC35 and SC35F viruses, respectively. Additionally, reporter constructs pPol-I-NP-Luc (encoding firefly luciferase) and pRL-TK (Promega; encoding Renilla luciferase) were co-transfected for normalization [10 (link)]. As a background control, vRNPs were transfected omitting the PB2 subunit. Cells were lysed using passive lysis buffer (Promega) and luciferase activity was determined 24 h post transfection using Dual-Luciferase Reporter Assay System (Promega) according to the manufactureŕs instructions and measured in a Tristar LB 941 Luminometer (Berthold).
7
Viral Gene Transduction Efficiency Assay
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The cells were exposed to ≈ 100 (HeLa and HaCaT) or ≈ 500 (NHEK) viral genome equivalents (vge) of PsVs per cell. One day after infection the cells were lysed with cell culture lysis reagent (Promega, Fitchburg, MA) and relative luciferase activity as gene transduction efficiency was measured using Luciferase substrate buffer (1 mM coenzyme A, 50 mM luciferin, 50 mM ATP, 0.5 M EDTA, 1 M DTT, 0.5 M Tris–HCl, pH 7.8, 1 M MgSO4) and normalized to lactate dehydrogenase (LDH) measurements (CytoTox-ONE Homogeneous Membrane Integrity Assay, Promega). Both luciferase and LDH activities were measured using the Tristar LB 941 luminometer (Berthold Technologies, Bad Wildbad, Germany).
8
Antiviral Activity Screening of Juncus acutus Against HCoV-229E
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Hepatoma cell line Huh-7 and Huh-7 cells transduced with a lentivirus encoding for TMPRSS2 protease, a cellular protease that allows fusion of the virus at the host cell surface (Huh-7/TMPRSS2), were used for all HCoV-229E infection assays. All the above-mentioned samples from Juncus acutus stems were screened for their antiviral activity against HCoV-229E-Luc expressing the luciferase, a recombinant HCoV-229E with luciferase reporter gene. Huh-7 cells seeded in 96-well plates were inoculated with HCoV-229E-Luc in the presence of each extract, fraction or compound for 1 h. Inoculum was then removed and replaced with culture medium containing the compounds for another 6 h. Finally, cells were lysed in 20 µL of Renilla lysis buffer (Promega) and luciferase activity was quantified using Renilla luciferase assay kit (Promega). Luciferase activity was measured by the use of a Tristar LB941 luminometer (Berthold Technologies, Bad Wildbad, Germany).
9
Quantifying β-Catenin Activity via Luciferase Assays
10
Mitochondrial Membrane Potential and ATP in HK-2 Cells
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To estimate changes in mitochondrial membrane potential, HK-2 cells were exposed to 0, 10, or 100 nM OTA for 48 h and were stained using JC-10 dye (Sigma-Aldrich, München, Germany) following the manufacturer’s instructions. A Cytation 3 Image Reader (BioTek, Berlin, Germany) was used for signal detection.
Additionally, ATP content of HK-2 cells was measured after 48 h incubation with 0, 1, 10, or 100 nM OTA using an ATP Bioluminescence Assay Kit HS II (Roche, Basel, Switzerland) following the manufacturer’s instructions. A TriStar LB941 Luminometer (Berthold Technologies, Bad Wildbad, Germany) was used for detection. Protein concentrations were determined after cell lysis using bicinchoninic acid (BCA) in order to normalize the amount of ATP obtained for each cell-culture well.
Additionally, ATP content of HK-2 cells was measured after 48 h incubation with 0, 1, 10, or 100 nM OTA using an ATP Bioluminescence Assay Kit HS II (Roche, Basel, Switzerland) following the manufacturer’s instructions. A TriStar LB941 Luminometer (Berthold Technologies, Bad Wildbad, Germany) was used for detection. Protein concentrations were determined after cell lysis using bicinchoninic acid (BCA) in order to normalize the amount of ATP obtained for each cell-culture well.
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