Anti-protein and anti-polysaccharide serum antibodies were detected by indirect ELISA as described previously (12 (link),13 (link)). Briefly, Microlon high binding plates (Greiner Bio-One) were coated overnight at 4°C with either 10 ug/ml ovalbumin (Sigma) or S. pneumoniae serotype 14 polysaccharide (kindly supplied by John Schreiber, Tufts University, Boston, MA) diluted in PBS. Serial dilutions of serum were used to probe antigen bound plates. Detection was performed using biotinylated anti-mouse IgG polyclonal antibody (Jackson ImmunoLabs) and europium-conjugated streptavidin (PerkinElmer) followed by quantification by time-resolved fluorescence on a Victor3V Multilabel Counter using DELFIA Enhancement Solution according to the manufacturer’s protocol (PerkinElmer).
Victor3 5 multilabel counter
Manufactured by PerkinElmer
Sourced in United States
The VICTOR3 V Multilabel Counter is a versatile instrument designed for fast and accurate detection of various types of assays. It provides a wide range of detection modes, including absorbance, fluorescence, luminescence, and time-resolved fluorescence, allowing users to perform a diverse set of applications in life science research and drug discovery.
Automatically generated - may contain errors
Lab products found in correlation
Lactate dehydrogenase activity assay kit, by Merck Group (2 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Europium conjugated streptavidin, by PerkinElmer (1 mentions)
Delfia enhancement solution, by PerkinElmer (1 mentions)
Reporter lysis buffer, by Promega (1 mentions)
Easycyte 5 ht instrument, by Merck Group (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Suc llvy amc, by Merck Group (1 mentions)
27 protocols using victor3 5 multilabel counter
1
ELISA-Based Serum Antibody Detection
2
Proteasome Activity Assay in Cells
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Determination of proteasome activity was accomplished by measuring hydrolysis of the fluorogenic peptide SUC-LLVY-AMC as previously described [21 (link)]. Thus, cells were cultivated in 96-well clear bottom black dish in phenol-red free DMEM. After settlement, cells were treated for 24 h with EGb 761 or vehicle. Then, culture medium was replaced with medium containing 100 μM SUC-LLVY-AMC with or without 10 μM MG132. Probes were incubated for 60 min and then lysed with reporter lysis buffer (Promega, USA). Turnover of SUC-LLVY-AMC was determined by measuring AMC fluorescence using Victor3V Multilabel counter (Perkin Elmer, USA) at 460 nm emission. Specific proteasome activity was calculated as the difference between total activity and activity in the presence of MG132.
3
Intracellular ROS Detection by DCFH-DA Assay
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For intracellular ROS detection, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used, as previously reported [27 (link)]. Pre-treated cells were incubated with 10 μM DCFH-DA in DMEM without phenol red for 30 min. After DCFH-DA removal, cells were incubated with 100 μM H2O2 in PBS for 15 min. Cells were washed with PBS and the fluorescence was measured using 485 nm excitation and 535 nm emission with a microplate spectrofluorometer (VICTOR3 V Multilabel Counter, PerkinElmer, Wellesley, MA, USA). Intracellular ROS were also evaluated at single cell level by flow cytometry. A number of 2 × 105 cells was seeded in 12-well tissue culture plates and left in culture overnight. The cells were treated with crocin or kaempferol (5 μM) for 1 h in DMEM 10% FBS at 37 °C 5% CO2, then stained with 10 μM DCFH-DA in DMEM without phenol red and FBS for 30 min, 37 °C 5% CO2. The cells were detached with trypsin solution and centrifuged at 300× g for 5 min, in 1.5 mL tubes. After removing the supernatant, the cell pellet was resuspended and appropriately diluted to 5 × 10−5 cells/mL in PBS. Guava® easyCyte™ 5 HT instrument was used to collect flow cytometry data. FlowJo software was used to analyse the geometric mean fluorescence intensity (MFI). Unstained untreated samples were used as controls.
4
Spinal Cord Cytokine Analysis in Rats
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On day 8, all of the rats were sacrificed following an overdose of pentobarbital anaesthesia. The L4–L6 left-half of the spinal cord was immediately removed and stored at -80 °C prior to further treatment. Frozen samples were directly homogenised in a buffer in the presence of protease inhibitors (Sigma). After centrifugation at 13,000 rpm for 20 min, the supernatant was obtained for IL-1, IL-6, TNF-α and IL-10 protein analysis. The Bradford protein assay was used to determine the total protein concentration. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to assess the cytokine proteins (R&D Systems, Minneapolis, MN, USA; sensitivity: 5 pg/ml). ELISA microplates were analysed using a Victor3 V multilabel counter (1420, Perkin-Elmer, Boston, MA, USA), and data were standardised as picograms of TNF-α, IL-1β, IL-6 and IL-10 per 200 μg of total supernatant protein. The concentration of each target cytokine was determined based on an appropriate set of internal standard curves using recombinant rat cytokines (n = 6 each group).
5
MTT Assay for Cell Viability
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AFSCs were seeded in 96-well plates at a density of 10000 cells/well (day 0) in 200 μl of a culture medium, 4 replicates for each condition. At the end of each experiment, 0.5 mg/ml MTT was added and incubated for 1.5 h at 37°C. After incubation, MTT solution was removed and DMSO was added to solubilize the formazan salts. The absorbance was measured at λ = 595 nm using a microplate spectrophotometer (VICTOR3 V Multilabel Counter; PerkinElmer, Wellesley, MA, USA).
6
Measuring Intracellular ROS in SH-SY5Y Cells
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The production of intracellular reactive oxygen species was evaluated using the fluorescent probe DCFH-DA as reported in [66 (link)]. Briefly, differentiated SH-SY5Y were treated with oleocanthal for 24 h and then incubated with 10 µM DCFH-DA in DMEM w/o FBS for 30 min. After DCFH-DA removal, cells were incubated with H2O2 for 30 min. Cells were washed with PBS and the fluorescence was measured using 485 nm excitation and 535 nm emission with a microplate spectrofluorometer (VICTOR3 V Multilabel Counter, PerkinElmer, Wellesley, MA, USA).
7
Transient Transfection Assay in MEFs
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For an ex vivo transient transfection assay, WT and Msh2−/− MEF cells were seeded in 12-well plates (50 000/well) and one day later were cotransfected with 75 ng of pCMVIG-derived plasmids and 75 ng of the pRL-CMV plasmid, which was used as an internal control of transfection efficiency, using the ExGen 500 reagent (Euromedex, Souffelwryersheim, France). Dual-reporter assays were performed 18 h after the transfection using the Dual-Glo Luciferase Assay System (Promega, France). Firefly and Renilla luminescence were measured in a VICTOR3 V Multilabel Counter (Perkin Elmer) and the data were processed in the Wallac 1420 Workstation software. In all the experiments, the transfection and luciferase assays were performed in triplicate. The firefly luciferase activity was normalized to the Renilla luciferase activity; for each pCMVIG-based construct, this relative luciferase activity was normalized to the firefly/Renilla ratio obtained with the unmodified pCMVIG plasmid. The final ratio was expressed as a fold change.
8
Oleocanthal Modulates Oxidative Stress
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Cells were treated with different concentrations of oleocanthal (1–10 µM) for 24 h prior to induction of oxidative stress. Cell viability was evaluated by measuring formazan formation as previously reported [64 (link),65 (link)]. Briefly, after treatments, cells were incubated with 0.5 mg/mL of MTT solution for 1 h at 37 °C. After incubation, MTT was removed and 100 µL of DMSO were added, and the absorbance was recorded at λ = 595 nm using a microplate spectrophotometer (VICTOR3 V Multilabel Counter; PerkinElmer, Wellesley, MA, USA).
9
Evaluating Cellular Oxidative Stress Response
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Cells were treated with different concentrations of plain and hill extracts (1 µg/mL–500 µg/mL) for 24 h. The induction of oxidative stress was achieved with 700 µM of H2O2 for 1 h. Cell viability was evaluated by measuring formazan formation as previously reported [41 (link)]. Cells were incubated with 0.5 mg/mL of MTT solution for 1 h at 37 °C. After incubation, MTT was removed and 100 µL of DMSO were added, the absorbance was recorded at λ = 595 nm using a microplate spectrophotometer (VICTOR3 V Multilabel Counter; PerkinElmer, Wellesley, MA, USA). Lactate dehydrogenase (LDH) activity was evaluated in the culture medium and the test was performed using the Lactate Dehydrogenase Activity Assay Kit according to the manufacturer’s instructions.
10
MTT Assay for Cell Viability
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In vitro cell viability of PRNCs and controls was measured using 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). CCRF-CEM or Ramos cells were counted and transferred to 96-well plates at a density of 8 × 103 cells per well in 80 μL serum containing media. Treatment of each samples were performed by adding 10 μL of PBS dispersed samples to the wells. After incubating 18 h, 10 μL of MTT solution (5 mg mL-1) was added in each wells. Cells were then lysed by adding 100 μL 10% SDS aqueous solution (pH = 5.5, Bioneer, Korea) then incubating overnight. The absorbance of formazan salt at 570 nm was measured using a microplate reader (VICTOR 3-V Multilabel counter, PerkinElmer, Wellesley, MA).
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