The assay was performed as in ref. [61 ]. Optical density at 570 nm (OD570) was measured by using Wallac Victor3 1420 Multilabel plate reader (Perkin Elmer). Experiment was performed in biological triplicates, where each biological replicate consisted of technical quadruplicates.
Wallac victor3 1420 multilabel plate reader
Manufactured by PerkinElmer
Sourced in Finland
The Wallac Victor3 1420 Multilabel plate reader is a versatile laboratory instrument designed for high-throughput multimode detection. It is capable of performing fluorescence, luminescence, and absorbance measurements on microplates.
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6 protocols using wallac victor3 1420 multilabel plate reader
1
Optical Density Assay Protocol
2
Viability Assay for Bovine Aortic Endothelial Cells
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Cells were plated at concentration of 4 × 10 4 cells/ mL in a 96-well plate and allowed to culture for 24 h before experimentation. Extract treatments were applied to BAEC cultures by replacing the base medium with culture media containing PMG, TA, CH, and GM treatments at 80 μg/mL with or without LPS exposure at 25 ng/mL and incubated for 12 h at 37°C. The BAEC were exposed to LPS as positive control and to a culture medium without any treatments as negative control. The LPS concentration was determined on the basis of cell viability, which was more than 80% compared with negative control.
The viability of BAEC was measured using the Promega Cell Titer-Glo Viability Assay (Promega Corp., Madison, WI), which quantifies the amount of ATP, an indicator of metabolically active cells. A mixture of substrate and buffer results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP directly reflects the number of viable cells. Luminescence was measured using a Wallac Victor3 1420 Multilabel Plate Reader (PerkinElmer Inc., Waltham, MA).
The viability of BAEC was measured using the Promega Cell Titer-Glo Viability Assay (Promega Corp., Madison, WI), which quantifies the amount of ATP, an indicator of metabolically active cells. A mixture of substrate and buffer results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP directly reflects the number of viable cells. Luminescence was measured using a Wallac Victor3 1420 Multilabel Plate Reader (PerkinElmer Inc., Waltham, MA).
3
Intracellular ROS Measurement in BAEC
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Cells were plated the day before assay at concentration of 4 × 10 4 cells/mL in 96-well plate. Treatments were applied as described above. The intracellular ROS production was measured with OxiSelect Intracellular ROS Assay Kit (Cell Biolabs, San Diego, CA). The flu-orescent probe chloromethyl-2,7-dichlorofluorescein diacetate diffuses through the cell membrane and is hydrolyzed by esterases to non-fluorescent dichlorofluorescein. In the presence of ROS, this compound is oxidized to highly fluorescent dichlorofluorescein. Briefly, BAEC were seeded in black 96-well plate overnight. The media were then changed to 0% FBS, 20 mM HEPES, 2 mM l-glutamine, 1% (vol/vol) antibiotic-antimycotic, heparin (100 μg/mL), insulin (10 μg/mL), transferrin (5 μg/mL), and sodium selenite (10 ng/mL) before the assay. Fluorescence was measured using a Wallac Vic-tor3 1420 Multilabel Plate Reader (PerkinElmer Inc., Waltham, MA).
4
Fluorescence-based Assay Measurements
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The assay was performed as in [59 (link)]. Measurements were done by using either a Wallac Victor3 1420 Multilabel plate reader (Perkin Elmer) or Synergy H1 Hybrid Multi-mode reader (BIOTEK). All experiments were done at least in biological quadruplicate, where each biological replicate consisted of technical duplicate.
5
Cell Proliferation Assay by MTT
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Cell proliferation was determined by the MTT assay (also referred as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). MTT cells (15×103) were incubated in 96-well plates for 24 hours in complete medium before the addition of the indicated compound. A solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (1 mg/ml; Sigma-Aldrich) was added and plates were incubated at 37°C for 3 hours before measuring absorbance at 562 nm using a Wallac Victor 3 1420 Multilabel plate reader (Perkin Elmer).
6
Quantifying Cellular Reactive Oxygen Species
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ROS were measured using the DCFDA/H2DCFDA-Cellular ROS Assay Kit (#ab113851, Abcam, Cambridge, UK). The assay is based on the use of 2′,7′ –dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA), a membrane-permeable fluorogenic dye that quantitatively measures hydroxyl, peroxyl, and other reactive oxygen species (ROS) in living cells. After diffusion into the cell, the DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2′,7′ –dichlorofluorescein (DCF). DCF is highly fluorescent and is detected by fluorescence spectroscopy.
Briefly, mouse bone marrow neutrophils were seeded into black clear bottom 96-well plates (100,000 cells/well) and stimulated as described above. For measurement, the medium was aspirated and cells were resuspended in 100-μL DCFDA solution (20 μM) and incubated for 30 min at 37 °C protected from the light. Thereafter, cells were carefully washed twice with the 1× Buffer solution provided in the kit and resuspended in 1 ×Supplementary Buffer (supplemented with 10% FCS). The levels of intracellular ROS were detected by the fluorescence spectroscopy with excitation (Ex)/emission (Em) at 485 nm/535 nm using a PerkinElmer Wallac Victor3 1420 Multilabel Plate Reader (PerkinElmer Life and Analytical Sciences, Turku, Finland).
Briefly, mouse bone marrow neutrophils were seeded into black clear bottom 96-well plates (100,000 cells/well) and stimulated as described above. For measurement, the medium was aspirated and cells were resuspended in 100-μL DCFDA solution (20 μM) and incubated for 30 min at 37 °C protected from the light. Thereafter, cells were carefully washed twice with the 1× Buffer solution provided in the kit and resuspended in 1 ×Supplementary Buffer (supplemented with 10% FCS). The levels of intracellular ROS were detected by the fluorescence spectroscopy with excitation (Ex)/emission (Em) at 485 nm/535 nm using a PerkinElmer Wallac Victor3 1420 Multilabel Plate Reader (PerkinElmer Life and Analytical Sciences, Turku, Finland).
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