Thiazolyl Blue Tetrazolium Bromide (MTT), 3, 3′, 5 Triiodothyronine and puromycin were obtained from Sigma (St. Louis, MO). MEM, RPMI 1640, DMEM, L-glutamine, penicillin/streptomycin, SeaPlaque™ Agarose was from Lonza (Walkersville, MD). Fetal bovine serum was from Gemini Bio Products, (West Sacramento, CA). Lipofectamine LTX, SuperScript® III Reverse Transcriptase and qPCR probes (ESR1, THRB, COUP-TF1 and GAPDH) were provided by Life Technologies (Grand Island, NY). The renilla luciferase assay kit was from Promega (Madison, WI). TRβ and GAPDH antibodies were from Santa Cruz Biotechnology (Dallas, TX). Total ERα antibody was from Vector Laboratories (Burlingame, CA). AR, c-PARP, PARP, c-Caspase 3, Caspase 3, pPKA and PKA antibodies were from Cell Signaling Technology (Beverly, MA). ChemiGlow Chemiluminescent Substrate kit was from Protein Simple (Santa Clara, CA). Docetaxel and doxorubicin were from LC Laboratories (Woburn, MA). Cisplatin was from ENZO (Plymouth Meeting, PA). c-AMP inhibitor bupivacaine and H89 were obtained from Selleck Chemicals (Houston, TX)..
Renilla luciferase assay kit
Manufactured by Promega
Sourced in United States
The Renilla Luciferase Assay Kit is a laboratory product designed to quantify Renilla luciferase activity. It contains reagents necessary to measure the bioluminescent signal generated by the Renilla luciferase enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Minimum essential medium (mem), by Lonza (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (2 mentions)
Puromycin, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Gemini Bio (2 mentions)
Seaplaque agarose, by Lonza (2 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (2 mentions)
Rpmi 1640, by Lonza (2 mentions)
Penicillin streptomycin, by Lonza (2 mentions)
L glutamine, by Lonza (2 mentions)
Dual luciferase reporter assay kit, by Promega (2 mentions)
Glomax 20 20, by Promega (2 mentions)
Euthasol, by Virbac (2 mentions)
Centro xs3 lb 960, by Berthold Technologies (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Cisplatin, by Enzo Life Sciences (1 mentions)
P pka, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Doxorubicin, by LC Laboratories (1 mentions)
34 protocols using renilla luciferase assay kit
1
Molecular Profiling of Nuclear Receptors
2
Minicircle Transfection for CHIKV Replication
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The minicircle forms of CMV-P1234 and MCC-P1234 were obtained as described [53 (link)]. These minicircles and all plasmid DNAs, used for transfection experiments, were purified using Nucleobond Xtra Midi EF kit (Macherey-Nagel). BHK-21, Huh7, U2OS or BSR cells, grown on 35 mm plates to 90% confluence, were co-transfected with mixtures consisting of 1 μg of plasmid encoding CHIKV replicase and 1 μg of plasmid for expression of corresponding RNA template using Lipofectamine LTX reagent according to the manufacturer's protocols (Invitrogen). COP-5 cells were grown and treated similarly and Lipofectamine 2000 (Invitrogen) was used as transfection reagent. At 18 h post transfection cells were lysed with Renilla luciferase assay lysis buffer (Promega) and Fluc and Gluc activities were analyzed using the Dual-Luciferase Reporter Assay kit and Glomax SIS luminometer (Promega); total amount of protein in cell lysates was measured using Bio-Rad protein assay system according to the manufacturer's protocols. In some experiments aliquots of growth media were collected at selected time points and Gluc activity was measured using Renilla Luciferase Assay kit (Promega).
3
Luciferase Reporter Assay for HCV Replicon
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The Luciferase reporter gene assay was performed, using the Renilla Luciferase assay kit (Promega, Madison, WI), according to the manufacturer’s protocol. Briefly, HCV subgenomic replicon cells, APC140, treated with HIV-1 pseudovirus or virions or transfected with the indicated plasmids for 48 hr, were washed twice with ice-cold PBS and lysed in Renilla luciferase lysis buffer. The luciferase activity in the lysates was determined with a luminometer (Promega) in triplicates of three independent experiments.
4
Dual Luciferase Reporter Assay in HEK 293T
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Dual luciferase reporter constructs (Firefly and Renilla, 1μg/well) were transfected into HEK 293T cells at 70% confluency in 48 well plate using lipofectamine 2000. Compounds were added for 16 hours, followed by luciferase assay using Dual Luciferase reporter assay kit (Promega, USA). Firefly luciferase values were divided by that of renilla and plotted. Gaussia luciferase was measured from culture medium using renilla luciferase assay kit (Promega, USA). Viability of same cells were measured using stable tetrazolium salt WST-1 (Roche, USA). Gaussia values were normalised to that of cell viability and plotted. Values are mean ± SEM of three independent experiments done in triplicate.
5
Investigating NF-κB Activation in HeLa Cells
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Cytokines: Human tumor necrosis factor-α (TNF-α); Human oncostatin M (OSM); Human interleukin-1β (IL-1β); Human leukemia inhibitory factor (LIF) (Sigma); IL-1 receptor antagonist (IL-1RA).
Reagents for genetic engineering: a lipofection reagent (TransIT-LT1, Mirus); a mammalian expression vector (pcDNA 3.1(+)); cDNA encoding p50 of NF-κB; cDNA encoding RLuc.
Immunocytochemistry-related reagents: mouse anti-Flag antibody (Sigma); mouse anti-NF-κB P50 antibody (Abcam); Cy-5-conjugated secondary antibody (Jackson); paraformaldehyde (PFA) (Sigma); fish skin gelatin (FSG) (Sigma); Mowiol solution (Aldrich); Sytox Green (Molecular Probes).
Buffers: a TBST buffer (Tris–HCl buffer with saline and Triton X-100); a phosphate buffered saline (PBS) buffer.
Cell culture and assay reagents: human uterine cervical carcinoma-derived HeLa cells; Dulbecco’s modified eagle’s medium (DMEM; Sigma); cholesterol-free fetal bovine serum (FBS; Gibco); penicillin-streptmycin (P/S); Renilla luciferase assay kit (Promega), a Bradford reagent (Pierce).
6
Neutralizing Activity Quantification of SARS-CoV-2 Variants
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To measure the neutralizing activity of sera or IgGs against various SARS-CoV-2 variant pseudotyped viruses, 1 × 104 293T-ACE2 cells (100 μL) were seeded in 96-well plates in DMEM low-sugar medium (Gibco). The next day, the sera from HR121-immunized animals were three-fold serially diluted in another 96-well plate in a volume of 60 μL. Then, 60 μL SARS-CoV-2 pseudovirus (M.O.I. = 0.1) was added to the diluted sera and incubated for 1 h at 37 °C. Thereafter, 100 μL of the mixture was incubated with 293T-ACE2 cells for 24 h at 37 °C, and the supernatant was removed from the cells after incubation. Renilla luciferase activity was determined using a Renilla luciferase assay kit (Promega, Madison, WI, USA). The NT50s of the sera or IC50s of the IgGs were calculated in GraphPad Prism 8.0.1 software as mentioned above.
7
Quantifying Transcriptional Response in Chlamydomonas
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Each transgenic C. reinhardtii sample in the mid-exponential phase was prepared with the same cell density based on absorbance (OD750nm = 1.0). The cells were incubated in the dark for 12 h to remove the effects of other stress stimuli, and then transferred to dark and low-temperature conditions (20°C, 10°C, or 0°C). After low-temperature treatment, cells were harvested by centrifugation at 13,000 ×g for 2 min. Luciferase assays were performed using a Renilla Luciferase Assay Kit (Promega, USA) with a modified protocol [15 (link)]. Cell pellets were resuspended in 100 μl of 1× lysis buffer and vortexed vigorously for 3 min. After centrifugation at 13,000 ×g for 5 min (4°C), 90 μl of supernatant and 10 μl of 1× luciferase substrate were mixed in a new 1.5 ml tube. Immediately after mixing, the luminescence was measured using a GloMax 20/20 (Promega).
8
Quantifying hCoV-229E Replication in Huh7 Cells
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To determine replication level of Renilla hCoV-229E, the supernatant of infected Huh7 cells was removed 24h postinfection and the cells lysed in 200μl of Renilla luciferase lysis buffer. Renilla luciferase of hCoV-229E was determined by Renilla Luciferase Assay Kit (Promega) according to the manufacturer’s instructions on an Orion microplate luminometer (Berthold).
9
Light-Inducible Luciferase Activity Analysis
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For light inducibility analysis, transgenic cells harboring the pCvpsaD::GLuc cassette were placed under various constant light conditions (dark, 80, 300, and 600 μmol photons m−2 s−1) for 2 h [29 (link)]. After the exposure to different light intensities, the cell density was adjusted to equal OD value (OD750nm = 0.5) and cells were then spun down by centrifugation (4000× g for 3 min). Luciferase activity was measured, according to the procedure described above using the Renilla Luciferase Assay Kit (Promega, Fitchburg, WI, USA). Light inducibility analysis was performed with two different transgenic lines.
10
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).
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