HeLa cells were seeded into 12-well plates (150,000 cells/well) and transfected with the indicated plasmids using polyethylenimine (PEI, Polysciences). Twenty-four hours after transfection cells were washed in 1 ml 1X PBS (Sigma) and lysed in 150 μl 1X Passive Lysis Buffer (Promega) for 10 min with rocking at room temperature. After lysis supernatants were cleared of debris by centrifugation at 10,000 x g for 1 min. For firefly luciferase, 40 μl of luciferase reagent (Promega) was added to 8 μl of sample, and luciferase activity was measured using a luminometer (Molecular Devices). For experiments using bicistronic reporters, 40 μl of Stop and Glo reagent was added to each sample after measuring firefly luciferase levels. 40 μl of renilla luciferase reagent (Promega) was then added, and luciferase activity was again measured. The amount of luciferase activity was normalized to the amount of protein present in each sample as determined by Bradford assay (Amresco).
Luciferase reagent
Manufactured by Promega
Sourced in United States
Luciferase reagent is a bioluminescent substrate used to detect and quantify the presence of luciferase, a light-producing enzyme. It serves as a versatile tool for various applications, including reporter gene assays, cell-based screening, and ATP detection.
Automatically generated - may contain errors
Lab products found in correlation
Passive lysis buffer (plb), by Promega (5 mentions)
Reporter lysis buffer, by Promega (5 mentions)
Lysis buffer, by Promega (4 mentions)
Glo lysis buffer, by Promega (3 mentions)
Glomax 96 microplate luminometer machine, by Promega (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
E1500, by Promega (2 mentions)
Pam3csk4, by InvivoGen (2 mentions)
Td 20 20, by Turner Designs (2 mentions)
β galactosidase β gal reagent, by Promega (2 mentions)
Cell culture lysis reagent, by Promega (2 mentions)
Spectramax m5 plate reader, by Molecular Devices (2 mentions)
Bradford protein assay, by Bio-Rad (2 mentions)
Genios plate reader, by Tecan (2 mentions)
Spectramax m5, by Molecular Devices (2 mentions)
Renilla luciferase reagent, by Promega (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Polyethylenimine (pei), by Polysciences (1 mentions)
Bradford assay, by Avantor (1 mentions)
Tgf β1, by Thermo Fisher Scientific (1 mentions)
50 protocols using luciferase reagent
1
Measuring Luciferase Activity in Transfected Cells
2
Transient Transfection and TGF-β Signaling
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Superfect® Transfection Reagent (Qiagen), was used for transient transfection according to the manufacturer’s instructions. A total of 1 μg from Luciferase Plasmid Reporters DNA [either wild type or mutated (−350/+150 ENG pXP2)] and 20 ng of pSV40-βgal reporter plasmid (for transfection normalization) were incubated in Opti-MEM (Gibco) with Superfect for 15 min at room temperature. The DNA-transfection reagent complexes were added to cells that were 80% confluent in P-24 wells. Transfection was allowed to proceed for 24 h at 37 °C and 5% CO2.
Cells were lysed in 1× lysis buffer from Promega. Relative units of luciferase (Promega luciferase reagent) were measured in a Glomax luminometer (Promega), and β-galactosidase units as measured by Galacto-light (Tropix) were used to normalize transfection efficiency. Each condition was repeated in triplicate. Treatments with TGF-β1 (Preprotech) proceeded for 24 h with 10 ng/ml. The control reporters of TGF-β, CAGA-luc, and BRE-luc were used in parallel as reporter controls of the TGF-β response.
Cells were lysed in 1× lysis buffer from Promega. Relative units of luciferase (Promega luciferase reagent) were measured in a Glomax luminometer (Promega), and β-galactosidase units as measured by Galacto-light (Tropix) were used to normalize transfection efficiency. Each condition was repeated in triplicate. Treatments with TGF-β1 (Preprotech) proceeded for 24 h with 10 ng/ml. The control reporters of TGF-β, CAGA-luc, and BRE-luc were used in parallel as reporter controls of the TGF-β response.
3
Transfection and Luciferase Assay for Entamoeba invadens
4
Luciferase Assay for Protein Quantification
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Luciferase assay protocol reported by Saher et al. [32 (link)] was followed with some modifications. Cells were washed with 1× PBS and then lysed with 25 µL of 0.1% Triton-X100 reagent. 5 µL of cell lysates were used to determine protein quantity using the DC Protein Assay (Bio-Rad, Hercules, CA, USA) protocol. The remaining 20 µL of the lysates were mixed with luciferase reagent (Promega, Stockholm, Sweden) automatically by the injector. The RLU of luciferase were determined (GloMax® 96 Microplate Luminometer machine-Promega, Stockholm, Sweden). The final results were represented as fold increase in luciferase activity, calculated by normalizing the RLU values to the total protein quantities and then further normalization against values of untreated cells.
5
Bioluminescence-Based Cytotoxicity Assay
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In order to establish target cells for bioluminescence-based cytotoxicity assay, a panel of cell lines were generated via lentiviral transduction including SW620-luciferase, SW620-HLA-A11-luciferase, CFPAC1-luciferase, CFPAC1-HLA-A11-luciferase, PANC-1-luciferase, and PANC-1-KRAS-G12V-luciferase. 20,000 target cells in T-cell medium were seed in 96-well-plate co-cultured with 1-2C, 3-2E TCR-T or mock-T cells at varying effector-to-target (E: T) ratios for 48 h in triplicate. Wells containing target cells with mock-T cells served as negative controls for baseline cytotoxicity. Subsequently, the supernatant was removed and the collected cells were treated with 50 μL 1 × lysis buffer (Promega, cat.E1941) on ice for 30 min. Then, 10 µL lysate supernatant were mixed with 50 µL Luciferase reagent (Promega, cat.E1501) in a LumaPlate‐96 white plate (Greiner, cat. 655074), followed by measuring BLI with a luminometer (Promega GloMax® 96 Microplate Luminometer). The % specific lysis of tumor cells was calculated using the formula: % specific lysis = (luminescence of tumor cell line cocultured with mock-T cells - luminescence of tumor cell line cocultured with TCR-T cells)/ luminescence of tumor cell line cocultured with mock-T cells × 100.
6
Cellular ATP Measurement in Fibroblasts
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Fibroblasts were seeded at the density of 1.5 × 104 cells/well in replicates of nines in 96-well tissue culture plates in growth medium incubated at 37 °C in 5% CO2. The next day cells were incubated in triplicates in DMEM containing 5 mM glucose, 4 mM glutamine, and 1 mM pyruvate (ATP baseline), or DMEM containing 4 mM glutamine, 1 mM pyruvate, and 5 mM 2-deoxy-D-glucose (2DG) to bock glycolysis (ATP 2DG), or DMEM containing 5 mM glucose, 4 mM glutamine, 1 mM pyruvate, and 1 μM oligomycin to block the mitochondrial ATPase (ATP Oligo). After 90 min incubation, cells were washed with phosphate buffered saline (PBS) and lysed in 30 μl tichloroacetic acid (2.5% W/V) on ice for 30 min. Following lysis, 20 μl aliquots were transferred into a separate plate for protein determination (DC Protein Assay). 45 μl Tris-acetate buffer (400 mM, pH = 8.0) was added to the remaining lysate. Cellular ATP content was measured after addition of 20 μl of luciferase reagent (Promega, Madison, WI) in a luminescence plate reader (Spectramax M5). Luminescence values were normalized against an ATP standard.
7
Neutralization Assay for COVID-19 Antibodies
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Neutralization activity was measured as described previously [30 (link)]. Briefly, mAb or plasma were serially diluted and incubated with pseudovirus in duplicate for 1 h at 37 °C. Cell control and virus control were set. Then, freshly trypsinized Huh-7 cells were added to each well. After 24 h of incubation at 37 °C with 5% CO2, 150 µL of the supernatant was removed from each well, and 100 µL of luciferase reagent (Promega, Madison, WI, USA) was added. Two minutes later, 150 μL of lysate was transferred to a black plate for the detection of luminescence using a Victor 3 luminometer (PerkinElmer, Waltham, MA USA). The 50% inhibitory dose or inhibitory concentration (ID50 or IC50) was calculated as the plasma dilution ratio or mAb concentration that caused a 50% reduction in relative luminescence units (RLU) compared with the level of the virus control subtracted from that of the control cell.
8
Luciferase Assay Protocol for Transfected Parasites
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Luciferase assays were performed in triplicate as described in [29] (link). Briefly, parasites were harvested 20–22 hours post-transfection and resuspended in lysis buffer supplemented with protease inhibitors (luciferase assay system, Promega E1500, 1× N-acetyl-L-leucyl-L-leucyl-L-argininal, 1× HALT protein inhibitor cocktail, 1× E-64). Protein concentration was determined by Bradford assay and luciferase activity was measured by a luminometer (Monolight 2010). 30 µg of total protein was added to luciferase reagent (Promega) and relative light units were recorded. For Renilla luciferase activity, the above protocol and the Dual-Luciferase® Reporter Assay System (Promega) protocol were used according to manufacturer's instructions to measure the Renilla luciferase after the Firefly luciferase.
9
NF-κB Activation Assay with TLR Stimulation
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Cells were transfected in a 12-well plate with 50 ng of an NF-κB-luciferase reporter plasmid and 450 ng of HA-tagged TLR15 plasmid or 225 ng of gagaTLR2B and 225 ng of gagaTLR1A plasmid (14 (link)). Twenty-four hours after transfection cells were redistributed into a 96-well plate. After 24-h cells were washed twice with DMEM without FCS and stimulated with: 100 ng/mL of Proteinase K (Sigma), Pam3CSK4, FSL-1 (both Invivogen) or 10 μL PMSF treated or untreated CANV culture supernatant in a total of 100 μL DMEM without FCS. After 5 h at 37°C cells were lysed in 50 μL reporter lysis buffer (Promega) at −80°C for 24-h. After thawing lysate was mixed with luciferase reagent (Promega) and luciferase activity was measured in a TriStar2 luminometer (Berthold). NF-κB activity is represented by luciferase activity in Relative Light Units (RLU). Results were expressed as fold increase in NF-κB activity of stimulated over unstimulated cells.
10
Luciferase Assay from Cell Lysates
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Lysates for luciferase assay were prepared in 1× passive lysis buffer (Promega), 100 μl per well of a 24-well plate. 10 μl of lysate was incubated with 50 μl luciferase reagent (Promega) and measured for 10 s using (Lumat LB9507, EG&G Berthold). Graphs are represent raw RLUs readings from three independent experiments.
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