The in vitro anti-inflammatory activity of the aqueous extract was evaluated using a cell model that has been previously described [55 (link)]. Briefly, R3/1-Nf-κB cells were seeded (5000 cells/well) in a white 96-well plate (BRANDplates®, cell grade). Cells were pretreated with different concentrations of the extracts (1, 10, 50, and 100 µg/mL) for 18 h in complete medium (DMEM 10% FBS, 1% L-glutamine, 1% Penicillin/Streptomycin). This process was followed by a 6 h stimulation with 10 ng/mL TNFα. To avoid components’ interference with the reading of the luciferase assay, cells were washed once with 100 µL of warm PBS before 50 µL of DMEM was added. Subsequently, 50 µL ONE-Glo™ Luciferase Assay Substrate (purchased from Promega Corporation, Madison, WI, USA) was directly added to the wells, followed by a luciferase measurement performed using a luminometer (Wallac Victor2 1420, Perkin-Elmer™ Life Science, Monza, Italy). Experiments were performed with biological and technical replicates and the results are shown as mean ± SD compared to untreated control cells. Statistical analysis was performed using one-way ANOVA with Bonferroni’s multiple comparisons test (p < 0.05 was considered significant). The cell viability for all the concentrations tested in the anti-inflammatory assay was verified by MTT assay on R3/1-Nf-κB cells.
Wallac 1420 victor2
Manufactured by PerkinElmer
Sourced in United States, Italy, Finland
The Wallac 1420 Victor2 is a multi-mode microplate reader designed for a wide range of applications in life science research. It is capable of performing various detection modes, including absorbance, fluorescence, and luminescence measurements. The instrument is equipped with a temperature-controlled incubator and shaker, allowing for precise control of experimental conditions.
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Lab products found in correlation
One glo luciferase assay substrate, by Promega (6 mentions)
Celltiter glo luminescent cell viability assay, by Promega (4 mentions)
Prestoblue, by Thermo Fisher Scientific (3 mentions)
Luciferase assay kit, by Promega (2 mentions)
Opti mem media, by Thermo Fisher Scientific (2 mentions)
Nf κb luciferase reporter, by Thermo Fisher Scientific (2 mentions)
Lipofectamine ltx plus, by Thermo Fisher Scientific (2 mentions)
Caspase glo 3 7 assay kit, by Promega (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Cell proliferation kit 1, by Roche (2 mentions)
Fluo 4 nw calcium assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Reporter lysis buffer, by Promega (1 mentions)
Psicheck 2 vector, by Promega (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
4 dimethylamino benzaldehyde, by Merck Group (1 mentions)
Gluteraldehyde, by Merck Group (1 mentions)
Hek293, by Signosis (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay reagent, by Promega (1 mentions)
82 protocols using wallac 1420 victor2
1
In Vitro Anti-Inflammatory Activity Evaluation
2
NF-κB Activation Assay in RAW 264.7 Cells
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RAW 264.7 cells were plated at 1 × 105 cells/well on 24-well culture plate and transiently transfected with NF-κB-luciferase reporter (Affymetrix Inc., Santa Clara, CA, USA) or pNF-κB-luciferase reporter (Stratagene California, La Jolla, CA, USA) using Lipofectamine® LTX & PLUS in OPTI-MEM media (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instruction. The transfected cells were treated with LPS (1 μg/mL) in the absence or presence of various concentrations of TECA, AST and TECA + AST for 24 hours. Reporter gene activity was assayed using the luciferase assay kit (Promega Co., Madison, WI, USA), measured by a luminescence counter (Wallac Victor2 1420; PerkinElmer Inc., Waltham, MA, USA).
3
Caspase 3/7 Activity Assay in Schistosomula
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The activity of caspase 3/7 was measured using the Caspase-Glo 3/7 assay kit (Promega) following the instructions. Cell lysates from schistosomula were obtained from 2,000 parasites cultivated in a 24-well plate with complete medium (as described above) and treated with MC3935 25 μM or vehicle (DMSO 0.25%). The luminescence was measured in a white-walled 96-well plate in a Wallac Victor2 1420 multilabel counter (PerkinElmer). The graphs were plotted using GraphPad Prism 8 software, and statistical significance was calculated with the Student´s t-test.
4
Luciferase Assay for NF-κB Activity
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After treatment, R3/1-NF-κBcells were washed twice with cold PBS followed by a freeze-thaw cycle with reporter lysis buffer (purchased from Promega Corporation, Madison, WI, USA) for complete cell lysis. After the freeze-thaw cycle, 100 µL ONE-Glo™ Luciferase Assay Substrate (purchased from Promega Corporation, Madison, WI, USA) was directly added to the wells, followed by a luciferase measurement performed using a luminometer (Wallac Victor2 1420, Perkin-Elmer™ Life Science, Monza, Italy).
5
Dual-Luciferase Assay for miRNA Targets
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Psi‐CHECK‐2 vector (Promega) containing both Renilla and firefly luciferase genes was used to clone JARID1B 3′UTR downstream of the Renilla gene (psi‐CHECK/Jarid1B). For transfection efficiency, the Renilla luciferase signal was normalized upon firefly signal. To perform luciferase assays, cells were seeded on 96‐well plates, co‐transfected with both luciferase containing plasmid (psi‐CHECK/Jarid1B and empty psi‐CHECK) and miR‐OE plasmids (pSP65/U1‐miR‐381‐3p, pSP65/U1‐miR‐486‐5p). Luciferase assays were performed 24 and 48 hours after transfection, using the Dual‐Luciferase Reporter Assay System (Promega), according to protocol guidelines. To evaluate firefly (reference) activity, 100 μL of Luciferase Assay Reagent II (LAR II) was added to each well and luminescence was measured using a Wallac VICTOR2 1420 (Perkin‐Elmer, Waltham, MA, USA) plate reader. Renilla luciferase activity was expressed relatively to firefly activity.
miRNA effect on psi‐CHECK/Jarid1B reporter expression was evaluated as a percentage of luciferase activity from pSP65/U1‐miR‐381‐3p or pSP65/U1‐miR‐486‐5p transfected cells compared to control vector (pSP65/U1) transfected cells. Data shown is the result of at least 4 independent experiments.
miRNA effect on psi‐CHECK/Jarid1B reporter expression was evaluated as a percentage of luciferase activity from pSP65/U1‐miR‐381‐3p or pSP65/U1‐miR‐486‐5p transfected cells compared to control vector (pSP65/U1) transfected cells. Data shown is the result of at least 4 independent experiments.
6
In vitro Cytotoxicity Assay for ITs
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In vitro efficacy of the ITs against the tumor cell line was assessed using CellTiter‐Glo® Luminescent Cell Viability Assay (Promega, cat# G7571, Madison, WI, USA) as described previously (Zheng et al.,2017 ). The cell viability assay measures the luminescence produced as a result of ATP production from metabolically active cells. The increasing concentrations of cytotoxic ITs lead to cell death and a corresponding reduction in ATP‐related fluorescence. The luminescence signals were recorded using Wallac Victor2 1420 multilabel counter (Perkin Elmer, Waltham, MA, USA).
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In vitro efficacy of the ITs against the tumor cell line was assessed using CellTiter‐Glo® Luminescent Cell Viability Assay (Promega, cat# G7571, Madison, WI, USA) as described previously (Zheng et al.,
7
MTT and LDH Cytotoxicity Assays
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The MTT reduction assay was performed 24 h after exposure to LPS as described earlier [23 (link)], with the following modifications. Briefly, following removal of the media, thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich) was added to cells at a concentration of 1.2 mM, after which the cells were incubated for 3 h at 37 °C and 5% CO2, until visible purple crystals were formed. Thereafter, media were removed, cells were lysed, and formazan crystals were dissolved with DMSO (Fisher Scientific, Loughborough, UK). Absorbances were read at 585 nm with a Wallac Victor2 1420 microplate reader (Perkin Elmer, Waltham, MA, USA). The results were calculated as a percentage of relative MTT reduction compared to the control wells. The Pierce LDH Cytotoxicity Assay Kit was used according to the manufacturer’s instructions (ThermoFisher Scientific). Briefly, assay reaction mixture was added to media samples collected 24 h after LPS exposure. After 30 min incubation in the dark, stop solution was added to each sample and the absorbances were measured at 490 nm. The results are presented as percentage of LDH release in relation to LDH positive control.
8
NRF2 Activation by Apple Polyphenols
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Thinned apple polyphenols (TAP) extract was evaluated for its ability to modulate the antioxidant response mediated by NRF2 activation using NRF2/ARE Responsive Luciferase Reporter HEK293 stable cell line (Signosis, Santa Clara, CA, USA) as previously described [46 (link)]. Briefly, HEK293 cells (10,000 cells/well) were treated with the extract (concentrations between 1 and 250 µg/mL). CDDO-Me 75 (bardoxolone methyl) 75 nM was used as a positive control [47 (link)]. After adding ONE-Glo™ Luciferase Assay Substrate (purchased from Promega Corporation, Madison, WI, USA) (100 µL/well), luciferase measurement was performed with a luminometer (Wallac Victor2 1420, Perkin-Elmer™ Life Science, Monza, Italy). Experiments were carried out with biological and technical replicates; values are reported as mean ± SD compared to untreated control cells. One-way ANOVA with Bonferroni’s multiple comparisons test (p < 0.05 was considered significant) was used for the statistical analysis.
9
NF-κB Activation Modulation by Nannochloropsis
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Cells were plated at 1 × 105 cells/well in a 24-well culture plate and transiently transfected with NF-κB-luciferase reporter (Affymetrix Inc., Santa Clara, CA, USA) or pNF-κB-luciferase reporter (Stratagene, Cedar Cree, CA, USA) using Lipofectamine LTX & PLUS (Invitrogen) in OPTI-MEM media (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The transfected cells were treated with LPS (1 μg/mL) in the absence or presence of various concentrations of Nannochloropsis oceanica for 24 h. The reporter gene activity was assayed using the luciferase assay kit (Promega Co., Madison, WI, USA), measured by a luminescence counter (Wallac Victor2 1420, PerkinElmer Inc., Waltham, MA, USA).
10
Measurement of IDO Activity in MSC Cultures
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The activity of IDO was determined by the measurement of L-kynurenine in the supernatant of four MSC cultures as described previously (22 (link)). Briefly, MSC were seeded at a density of 100,000 cells/well in a 6 wells plate and cultured for 4 days with or without 50 ng/mL IFN-γ. 30% trichloroacetic acid was added to the supernatant in a 1:3 ratio. Samples were incubated for 30 min at 50°C and spun down at 12,000 rpm for 5 min. Samples were plated in a 96 wells flat bottom plate and diluted 1:1 in Ehrlich reagent [200 mg 4-dimethylaminobenzaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in 10 ml of glacial acetic acid]. Absorbance was read at 490 nm using a Wallac Victor2 1420 multilabel plate reader (Perkin Elmer, Waltham, MA, USA).
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