MAPKs (ERK1/2, JNK, and p38) activation was evaluated using non‐competitive sandwich ELISA (Biolegend e‐Bioscience DX Diagnostic, MA), according to the manufacturer's instructions. BV‐2 cells (1 × 105 cells/well) were seeded into 24‐well plates and cultured for 24 hr. Cells were pre‐treated for 4 hr with CSE (1 μg/ml) then stimulated with LPS (250 ng/ml) for 30, 60, and 120 min, and lysed with 100 μl of the provided lysis buffer. The total protein content of each sample was evaluated by Bradford colorimetric method (Merck KGaA; Germany), using bovine serum albumi (BSA) (Merck KGaA, Germany) as a reference standard. Absorbance was measured at 450 nm using an MP96 microplate reader spectrophotometer (Safas, Monaco). The activation of MAPKs was calculated as the ratio of phosphorylated to total proteins, normalizing values to the untreated control. Three independent experiments were performed.
Mp96 microplate reader spectrophotometer
Manufactured by Safas
Sourced in Monaco
The MP96 is a microplate reader spectrophotometer that can measure absorbance in microplates. It is capable of measuring a wide range of wavelengths and supports various microplate formats.
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10 protocols using mp96 microplate reader spectrophotometer
1
Evaluating MAPK Activation in BV-2 Cells
2
Cytokine Production in BV-2 Cells
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BV‐2 cells (1 × 105 cells/well) were seeded into 24‐well plates and cultured for 24 hr. Cells were pre‐treated for 4 hr with CSE, CBD, or CAR (1 μg/ml) and then stimulated with LPS (250 ng/ml) for 2 hr. TNF‐α, IL‐6, and IL‐1β production were evaluated in BV‐2 cell lysate and medium together according to the manufacturer's instruction by non‐competitive sandwich ELISA (Biolegend e‐Bioscience DX Diagnostic, CA) dosages, as previously reported (Governa et al., 2019 (link)). Absorbance was measured at 450 nm using an MP96 microplate reader spectrophotometer (Safas, Montecarlo). Samples were assayed in duplicate. Dosages were performed in three independent experiments.
3
DPPH Radical Scavenging Assay of CSE
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The antiradical capacity of CSE was tested through 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) assay, performing the method previously optimized by Biagi et al. (2019 (link)). The method is based on the absorbance measurement (515 nm) of the non‐radical form of DPPH (namely DPPH‐H) that is yielded by the reduction reaction of an alcoholic solution of DPPH in the presence of a hydrogen‐donating antioxidant. Tested samples were dissolved in ethanol 96% vol/vol. Different sample concentrations (range 6.25–0.19 mg/ml) were mixed with DPPH (1:19) and, after incubation for 30 min at RT in the dark, the absorbance was read at 515 nm using an MP96 microplate reader spectrophotometer (Safas, Monaco). IC50 values were calculated using linear regression analysis. Three independent replicates were performed.
4
Cytokine Quantification in Murine Plasma
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Mice were put under general anesthesia by intraperitoneal injection of zoletil and xylazine cocktail. Blood samples were taken from the ventricle with a standard cardiac puncture method [22 ], centrifuged at 3000 × g for 10 min at 4 °C, and the plasma was collected. TNF-α, IL-1β, and IL-17 protein levels were evaluated by using noncompetitive sandwich ELISA (Biolegend e-Bioscience DX Diagnostic, Monza, Italy), following the supplier instructions. The absorbance was measured at 450 nm using a MP96 microplate reader spectrophotometer (Safas, Monaco), and cytokine levels were expressed as picograms per milliliter according to a standard calibration curve.
5
Cytokine Levels Assessment in Mice
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Mice were put under general anesthesia by intraperitoneal injection of zoletil and xylazine cocktail. Blood samples were taken from the ventricle with a standard cardiac puncture method, centrifuged at 3000 × g for 10 min at 4 °C, and the plasma was collected. TNF-α, IL-1β, and IL-17 protein levels were evaluated by using noncompetitive sandwich ELISA (BioLegend e-Bioscience DX Diagnostic, Monza, Italy), following the supplier’s instructions. The absorbance was measured at 450 nm using a MP96 microplate reader spectrophotometer (Safas, Monaco), and cytokine levels were expressed as picograms per milliliter according to a standard calibration curve.
6
Cell Viability Assay with CCK-8
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Cell viability was assessed using the cell counting kit 8 (CCK-8, Sigma-Aldrich) as previously described [28 (link)]. Briefly, 5 × 104 cells/well were seeded into 96-well plates and grown to confluence. Medium was then replaced with 100 µL of fresh DMEM containing increasing concentrations of the samples to the appropriate well. After the incubation time, 10 µL of CCK-8 was added to each well and incubated for 1 h. Absorbance was measured at 450 nm using a MP96 microplate reader spectrophotometer (Safas, Monte Carlo, Principality of Monaco). Treatments were performed in sextuplicate in three independent experiments and cell viability was calculated by normalizing the absorbance of the test wells to the untreated control.
7
Cell Viability Assay using CCK-8
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Cell viability was performed using a Cell Counting Kit (CCK-8, Sigma-Aldrich) according to the manufacturer’s instructions. A total of 5 × 105 cells/well were seeded into 96 multi-well plates and grown to confluence. The absorbance was measured at 450 nm using a MP96 microplate reader spectrophotometer (Safas, Monte Carlo, Principality of Monaco). The treatments were performed in six replicates in three independent experiments, and the cell viability was calculated by normalizing the values to the control’s mean.
8
Cell Viability Assay with CCK-8
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Cell viability was assessed by using CCK-8 (Sigma-Aldrich) assay, following the supplier instructions. 5x10 3 cells/well were seeded into 96-well plates and grown to confluence. Medium was then replaced with 100 µl of fresh RPMI containing treatments to the appropriate well. After the incubation time, 10 µl of CCK-8 was added to each well and incubated for 1 h. Absorbance was measured at 450 nm using a MP96 microplate reader spectrophotometer (Safas, Monte Carlo, Principality of Monaco). Treatments were performed in sextuplicate in three independent experiments and cell viability was calculated by normalizing values to the untreated control.
9
Evaluating IL-6 Production via ELISA
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The effect of the treatments on IL-6 production was evaluated by using non-competitive sandwich ELISA (Biolegend e-Bioscience DX Diagnostic, Monza, Italy), following the supplier instructions, as previously reported (Governa and Biagi, 2019) . 1x10 5 cells/well were seeded into 24-well plates and grown to confluence. Medium was then replaced with 1000 µl of fresh RPMI containing treatments to the appropriate well. After 2 h, cells were lysed by freezing (-80 °C) and thawing (+24 °C) samples three times. Absorbance was measured at 450 nm using a MP96 microplate reader spectrophotometer (Safas). Treatments were performed in triplicate and cytokines production was dosed in duplicate (n=6), by normalizing values to the untreated control.
10
Evaluation of MAPK Phosphorylation
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The phosphorylation of p38, ERK and JNK was also evaluated by using non-competitive sandwich ELISA (Biolegend e-Bioscience DX Diagnostic, Monza, Italy), following the supplier instructions. 1x10 5 cells/well were seeded into 24 multiwell plates and grown to confluence. Medium was then replaced with 1000 µl of fresh RPMI containing treatments to the appropriate well. After 1 h, cells were washed three times with PBS and lysed using 100 µl of lysis buffer supplied with the kit. Total protein content of each sample was evaluated by Bradford (Sigma-Aldrich) method, using albumin (Sigma-Aldrich) as a reference standard.
Absorbance was measured at 450 nm using a MP96 microplate reader spectrophotometer (Safas). Treatments were performed in triplicate and the phosphorylation rate of p38, ERK and JNK was calculated as the ratio between the phosphorylated and the total form, normalizing values to the untreated control.
Absorbance was measured at 450 nm using a MP96 microplate reader spectrophotometer (Safas). Treatments were performed in triplicate and the phosphorylation rate of p38, ERK and JNK was calculated as the ratio between the phosphorylated and the total form, normalizing values to the untreated control.
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