Gastric epithelial cells were transiently transfected with the reporter plasmid NF-κB-Luc [29 (link)], which contains three κB responsive elements controlling the luciferase gene. AGS cells were transfected by the calcium phosphate method, whereas GES-1 cells were transfected using Lipofectamine® (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The day after, the cells were treated for 6 hrs with the MIX extracts and TNFα (10 ng/mL), as previously described. Then, cell lysis and the luciferase enzymatic reaction were conducted through BriteliteTM Plus reagent (PerkinElmer Inc., Milan, Italy), according to the manufacturer’s instructions. The results (mean ± SD of at least three experiments) were expressed as relative% with respect to the luminescence of the pro-inflammatory conditions (100%).
Britelite plus reagent
Manufactured by PerkinElmer
Sourced in United States, Italy
BriteLite Plus Reagent is a laboratory product manufactured by PerkinElmer. It is designed to facilitate luminescent reactions for various analytical applications. The core function of the reagent is to generate a luminescent signal, which can be detected and measured using appropriate laboratory equipment.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (4 mentions)
Victor x3 multilabel plate reader, by PerkinElmer (3 mentions)
Lipofectamine, by Thermo Fisher Scientific (2 mentions)
Victor x3, by PerkinElmer (2 mentions)
Victor3, by PerkinElmer (1 mentions)
Synergy htx, by Agilent Technologies (1 mentions)
Milli q direct 8 system, by Merck Group (1 mentions)
Prestoblue cell viability assay, by Thermo Fisher Scientific (1 mentions)
Cell counting kit cck 8, by Merck Group (1 mentions)
Milli q water, by Merck Group (1 mentions)
Prism 9, by GraphPad (1 mentions)
Synergy neo2 multi mode assay microplate reader, by Agilent Technologies (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Polybrene, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Prism 6, by GraphPad (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Synergy neo2, by Agilent Technologies (1 mentions)
54 protocols using britelite plus reagent
1
NF-κB Activation in Gastric Cells
2
Evaluating NF-κB Transcriptional Activity
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Before transfection, the medium of HaCaT cells was replaced with a serum-depleted culture medium. Then, cells were transiently transfected with a reporter NF-κB-Luc plasmid (250 ng per well), containing the luciferase gene under the control of the E-selectin promoter characterized by three κB responsive elements, using Lipofectamine® 3000 Transfection Reagent (Invitrogen®; Thermo Fisher Scientific, Monza, Italy). The plasmid NF-κB-Luc was a gift from Dr. N. Marx (Department of Internal Medicine-Cardiology, University of Ulm; Ulm, Germany). The day after (16 h from transfection), cells were treated with C. acnes or TNF-α as previously stated. At the end of the treatment, the amount of luciferase produced in the cells was assessed using BriteliteTM Plus reagent (Perkin Elmer, Milano, Italy) according to the manufacturer’s instructions. The luminescence derived from the reaction between luciferase and luciferin was measured with a multiplate reader (Victor ×3; PerkinElmer, Milano, Italy). The results (mean ± SEM of at least three experiments) were expressed as a percentage relative to the stimulated control, which was arbitrarily assigned the value of 100%.
3
NF-κB and IL-8 Promoter Assays
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GES-1 and AGS cells were transiently transfected in 24-well plates with two different reporter plasmids, the NF-κB-Luc (50 ng/well) or the IL-8-Luc (100 ng/well) [29 (link)]. The NF-κB-Luc contains the luciferase gene under control of the E-selectin promoter characterized by three κB responsive elements, while IL-8-Luc contains the luciferase gene under control of a fragment of the native promoter of the human IL-8. The plasmid NF-κB-Luc was a kind gift of Dr. N. Marx (Department of Internal Medicine-Cardiology, University of Ulm, Ulm, Germany)m whereas the native IL-8-Luc promoter was kindly provided by Dr. T. Shimohata (Department of Preventive Environment and Nutrition, University of Tokushima Graduate School, Tokushima, Japan). GES-1 cells were transfected using Lipofectamine® (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), whereas AGS cells by the calcium phosphate method. Sixteen hours later, the cells were treated for 6 h with extracts in the presence of the pro-inflammatory mediators (TNFα 10 ng/mL). Six hours later, cells were harvested and the luciferase assay was carried out using the BriteliteTM Plus reagent (PerkinElmer Inc., Waltham, MA, USA), according to the manufacturer’s instructions. The results (mean ± SD of at least three experiments) were expressed as percentage, relative to stimulated control, which was arbitrarily assigned the value 100%.
4
Evaluating NF-κB-driven Transcription
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NF-κB-driven transcription was evaluated as previously reported [21 (link)]. In brief, Caco-2 cells were transiently transfected with a reporter plasmid responsive to NF-κB (250 ng per well), containing the luciferase gene under the control of the E-selectin promoter characterized by three κB responsive elements. Lipofectamine® 3000 Transfection Reagent (Invitrogen®; Thermo Fisher Scientific, Monza, Italy) was used for transfection assays. The plasmid was a gift from Dr. N. Marx (Department of Internal Medicine-Cardiology, University of Ulm; Ulm, Germany). BriteliteTM Plus reagent (Perkin Elmer, Milano, Italy) was used to assess the amount of luciferase produced into the cells, according to the manufacturer’s instructions. A VICTOR X3 Multilabel Plate Reader (Perkin Elmer, Milano, Italy) was used to measure the luminescence deriving from the reaction between luciferin and luciferase. The results (mean ± SEM of at least three experiments) were expressed as percentage relative to stimulated control, to which was arbitrarily assigned the value of 100%.
5
NF-κB and IL-8 Promoter Regulation
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HaCaT cells were plated in 24-well plates and transiently transfected with the plasmid NF-κB-LUC or native IL-8-LUC, both at 250 ng per well, using Lipofectamine® 2000, according to previous studies [32 (link)]. Sixteen hours later, the cells were treated for 6 h with increasing concentrations of VVWE in the presence of the pro-inflammatory mediators (TNF-α at 10 ng/mL or LPS at 5 μg/mL). After six hours, cells were harvested and the luciferase assay was performed using the BriteliteTM Plus reagent (PerkinElmer Inc., Massachusetts, USA) according to the manufacturer’s instructions. Data were expressed considering 100% of the luciferase activity related to the cytokine-induced promoter activity.
6
NF-κB Activity Modulation in HaCaT Cells
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HaCaT cells were cultured in 24-well plates for 48 h, and then transiently transfected with a reporter plasmid responsive to NF-κB (250 ng per well), containing the luciferase gene under the control of the E-selectin promoter characterized by three κB-responsive elements. Lipofectamine® 3000 transfection reagent (Invitrogen®, Waltham, MA, USA; Thermo Fisher Scientific, Monza, Italy) was used for the transfection assays. The plasmid was a gift from Dr. N. Marx (Department of Internal Medicine-Cardiology, University of Ulm; Ulm, Germany). A few day later, the cells were treated with TNF-α/IFN-γ or TNF-α/IL-4, in addition to HVE (5–125 μg/mL) or HT (1–10 μM) for 6 h. At the end of the treatment, the amount of luciferase produced into the cells was assessed using the BriteliteTM Plus reagent (Perkin Elmer, Milano, Italy), according to the manufacturer’s instructions. The luminescence deriving from the reaction between luciferase and luciferin was measured with a VICTOR X3 multilabel plate reader (Perkin Elmer, Milano, Italy). The results (mean ± SEM of at least three experiments) were expressed as a percentage relative to the stimulated control, which was arbitrarily assigned to a value of 100%. S6I (50 nM) and apigenin (20 μM) were used as reference inhibitors of TNF-α/IL-4 stimulation, while EGCG (20 μM) was used as a reference inhibitor of TNF-α/IFN-γ stimulation.
7
Luciferase Assay for NF-κB Activation
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HaCaT and HDF cells were seeded in 24-well plates (DB Falcon TM ) at the density of 60000 cells/well for 72 hours. Following, the cells were transiently transfected, by lipofectamine method, with a reporter plasmid containing luciferase gene under control of a promoter characterized by the presence of three responsive elements κB (NF-κB-LUC 250 ng/well). The plasmid NF-κB-LUC was a gift of Dr. N. Marx (Department of Internal Medicine-Cardiology, University of Ulm, Ulm, Germany). After overnight incubation, the cells were treated with CSE or CBD and TNFa 10 ng/mL for 6 hours.
Luciferase produced into the cells was assessed using Britelite TM Plus reagent (PerkinElmer, Walthman, MA, USA) according to the manufacturer's instructions. The luminescence derived from the reaction between luciferase and luciferin was read through a spectrophotometer (Victor X3, PerkinElmer, United States). The results are expressed as mean ± s.d. of at least three experiments. EGCG (20 µM) was used as reference compound.
Luciferase produced into the cells was assessed using Britelite TM Plus reagent (PerkinElmer, Walthman, MA, USA) according to the manufacturer's instructions. The luminescence derived from the reaction between luciferase and luciferin was read through a spectrophotometer (Victor X3, PerkinElmer, United States). The results are expressed as mean ± s.d. of at least three experiments. EGCG (20 µM) was used as reference compound.
8
Evaluating NF-κB Driven Transcription
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To evaluate the NF-κB driven transcription, AGS cells were plated in 24-well plates (30,000 cells per well). After 48 h, cells were transiently transfected by the calcium-phosphate method with the reporter plasmid (NF-κB-LUC, 50 ng/well) containing the luciferase gene under control of three κB responsive elements. The plasmid NF-κB-LUC was a gift of Dr. N. Marx (Department of Internal medicine-Cardiology, University of Ulm, Ulm, Germany). After 16 h, the cells were treated with the stimulus (TNFα 10 ng/mL) and the extract for 6 h. Curcumin (10 µM) was used as the reference inhibitor. At the end of this time, cells were harvested and the luciferase assay was performed using the Britelite TM Plus reagent (PerkinElmer Inc., Walthman, MA, USA) according to the manufacturer's instructions. Data were expressed considering 100% of the luciferase activity related to the cytokineinduced promoter activity.
9
HIV-1 Pseudovirus Infection Assay
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TZM-bl and TZM-bl/FcγRI cells with Tat-regulated luciferase reporter gene expression were used for quantification of viral infection and antibody neutralization as described previously (25 (link), 26 (link), 98 (link)). Briefly, 5 × 103 TZM-bl or TZM-bl/FcγRI cells were seeded overnight in white-walled 96-well plates at 37°C in a humidified atmosphere with 5% CO2. The next day, the medium was aspirated without disturbing the cells and mixtures containing HIV-1-pseudotyped lentivirus, DEAE dextran (10 μg/mL), and anti-gp41 antibodies were added to the cells. After incubation for 48 h, cells were lysed and luciferase activity was determined using britelite plus reagent (Perkin Elmer). Relative luminescence unit (RLU) values were quantified using a Synergy HTX multimode reader (BioTek), and percent infection calculated as described previously (34 (link)).
10
Virus Neutralization Assay Protocol
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Virus neutralization assays were done using single cycle HIV-1 pseudovirus infections of TZM-bl cells as described elsewhere [42 (link),45 (link),46 (link)]. Briefly, heat inactivated rabbit sera (56°C for 1hr), hybridoma supernatant or purified IgG was diluted in 100 μl of cell culture media (DMEM supplemented with 10% heat inactivated FBS and 1% penicillin/streptomycin). Test samples were diluted over a range of 1:20 to 1:43740 in cell culture medium and pre-incubated with virus (~150,000 relative light unit equivalents) for 1 hr at 37°C before addition of cells. Following a 48 hr-incubation, cells were lysed and Luc activity determined using a microtiter plate luminometer and BriteLite Plus Reagent (Perkin Elmer). Neutralization titers are the sample dilution (for serum) or antibody concentration (for sCD4, purified IgG preparations and monoclonal antibodies) at which relative luminescence units (RLU) were reduced by 50% compared to RLU in virus control wells after subtraction of background RLU in cell control wells.
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