101 L cells were seeded in triplicate at 2.2 × 104 cells/well on a clear 48-well tissue culture plate in phenol red-free DMEM with 5% charcoal-stripped FBS. After 24 hours, media were removed and replaced with media containing 0.1% dimethyl sulfoxide (DMSO) or a range of AF doses (100nM, 500nM, 1 μM, 10 μM). After 18 hours of compound treatment, the cells were washed with 50 μL 1× PBS (Gibco, Invitrogen) and lysed with 50 μL Tropix lysis buffer (100 mM K2HPO4, 0.2% Triton X-100, pH 7.8, Applied Biosystems). Cell lysate was mixed 1:1 with luciferase substrate (Promega, Madison, WI), and luminescence was measured with a 700-nm filter on a Victor X5 microplate reader (PerkinElmer, Waltham, MA). The Bradford method (Bio-Rad) was used to measure total protein in each sample. Raw luciferase data was normalized to both total protein and background luciferase expression in the DMSO control samples and expressed as fold-increase over DMSO.
Victor x5 microplate reader
Manufactured by PerkinElmer
Sourced in United States
The Victor X5 is a microplate reader that can be used to measure various types of assays in multi-well microplates. It is capable of absorbance, fluorescence, and luminescence detection across a wide range of wavelengths.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin coated plates, by PerkinElmer (2 mentions)
Delfia enhancement solution, by PerkinElmer (2 mentions)
Luciferase substrate, by Promega (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Tropix lysis buffer, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Evos fluorescence microscope, by Thermo Fisher Scientific (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Calcein am, by Thermo Fisher Scientific (1 mentions)
Evos m7000 microscope, by Thermo Fisher Scientific (1 mentions)
Atplite 1step luminescence assay system, by PerkinElmer (1 mentions)
Eu n1 labeledanti 6x his antibody, by PerkinElmer (1 mentions)
14 protocols using victor x5 microplate reader
1
Luciferase Assay for Compound Screening
2
Evaluating Ex-4 DMN Cytotoxicity
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The cytotoxicity of Ex-4 DMNs was evaluated via a MTT cell proliferation assay. HEK293T cells were cultured for 24 h in 3 ml of Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% foetal bovine serum (FBS) and 1% antibiotics, at a density of 105 cells/well in 6-well plates, at 37 °C, in a humidified atmosphere of 5% CO2. After treating HEK293T cells with 200 μl solutions of blank DMN and Ex-4 DMN dissolved in 200 μl PBS, the cells were incubated for 24 h. Cells were then incubated with 300 μl of MTT reagent (1 mg/ml) for 1 h, then the medium was removed, and the cells were treated with 1 ml of dimethyl sulfoxide (DMSO) per well. Cell viability (%) was calculated at the absorbance of 560 nm using a Victor X5 microplate reader (Perkin Elmer, Waltham, MA, USA). The viability of the PBS-treated control cells was set as 100%.
3
Artemisinin and TSA Cytoprotection Assay
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D407 cells (5 × 103 cells/well) and primary human RPE cells (1 × 104 cells/well) were applied and cultured in the 96-well plate. On the second day, the cell cultures were exposed to H2O2 after pretreatment with or without Artemisinin or TSA diluted in the FBS-free medium. After H2O2 treatment (500 μM) for 24 h, the cell cultures were incubated with 0.5 mg/mL MTT for 3 h. Thereafter, the formazan crystals formed and were dissolved with dimethyl sulfoxide (DMSO) (100 μL/well). The absorbance was detected at 570 nm by Victor X5 Microplate Reader (PerkinElmer, Waltham, MA, USA) to measure the cell viability. Results were normalized to the untreated control and expressed as a percent.
4
Cell Proliferation Assay by MTT
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Cell proliferation was measured by MTT assay. Cells (5 × 103/well) were seeded in 96-well plates in 100 μl DMEM. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (Sigma-Aldrich) solution (20 μl per well, 5 mg/ml in PBS) was added to each well. After incubation at 37°C for 2 h, the supernatant was removed. Formazan was dissolved in DMSO and absorbance was measured using the 540 nm filter of a Victor X5 microplate reader (Perkin Elmer, Waltham, MA).
5
Exendin-4 and Carboxymethylcellulose Interaction
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To analyse the interaction of CMC and Ex-4, 10 mg/ml of Ex-4 was mixed with the 4, 8, 10, and 12% (w/v) CMC polymer, and examined by a Vertex70 FT-IR spectroscope (Bruker, Billerica, MA, USA). All the samples were measured using potassium bromide (KBr) pellets. Transmittance spectra were acquired in the range of 400–4000 cm−1 by accumulating 32 scans at a resolution of 4 cm−1. The bioactivity of Ex-4 was measured using an ELISA assay kit according to the manufacturer’s instructions. The mixture solutions of CMC and Ex-4 were diluted at the concentrations of 0.01 to 100 ng/ml, and the absorbance was calculated using a Victor X5 microplate reader (Perkin Elmer, Waltham, MA, USA) at 560 nm.
6
EphA2-LBD Binding Assay Protocol
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Example 7
EphA2-LBD and EphA4-LBD were expressed and purified as we previously reported (30). Isothermal Titration calorimetry (ITC) measurements were obtained with a TA Instruments micro-calorimeter. The optimized DELFIA (Dissociation-Enhanced Lanthanide Fluorescent Immunoassay) assay protocol included a 2 h incubation of 100 μL of 1 μM 123B9-biotin in 96-well streptavidin-coated plates (PerkinElmer), followed by 3 washing steps. Subsequently, after pre-incubation of test agents for 15 min with a solution 0.712 μM EphA2-LBD, 11 μL of this mixture was added to 89 μL solution containing 4.17 nM Eu-N1-labeled anti-6×His antibody (PerkinElmer) and incubated for 1 h. After washing steps, 200 μL of the DELFIA enhancement solution (PerkinElmer) was added to each well followed by a 10-min incubation, prior to fluorescence reading (VICTOR X5 microplate reader, PerkinElmer; excitation and emission wavelengths of 340 and 615 nm, respectively). Fluorescence readings were normalized to that of DMSO control and reported as percent inhibition. The IC50 values were analyzed using GraphPad Prism Version 7 and data fitted with a nonlinear regression (least squares ordinary fit) of the log[compound] versus the observed response.
7
Inhibition Profiling of Compounds on Matrix Metalloproteinases
8
Anti-Cas9 Antibody ELISA Assay
9
Titration of SARS-CoV-2 Pseudovirus
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Titration of pseudovirus preparations followed a previously described protocol25 (link). Here, 104 293 T-ACE2 cells were seeded in 100 µl of DMEM10 into 96-well black/clear bottom plates purchased from ThermoFisher (catalog # 165,305). The infection was monitored by visualization and quantification of GFP+ cells using an EVOS fluorescence microscope (ThermoFisher). For titration, 2 × serial dilutions of the pseudovirus preparation were tested onto 104 293 T-ACE2 cells/well. Hundred microliters of diluted pseudovirus preparations were added to corresponding wells. Control (background) wells received 100 µl of DMEM10. After a 72 h-infection period, cells were harvested using trypsin treatment. Infectivity was quantified by detection of GFP+ cells using a BD FACSAria flow cytometer (BD Biosciences; Supplementary Fig. S2 ). Pseudovirus infectivity was also quantified by luciferase assay using duplicate wells. For luciferase assay readout, we opted for the previously described in-house luciferin buffer25 (link),26 (link). Assay plates were read using a Victor X5 microplate reader (Perkin Elmer).
10
Calcein-AM-based NK Cell Cytotoxicity Assay
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Ten thousand Calcein-AM (ebioscience, 65-0853-78, 5 µM)-labeled target cells (MSCs or K562 cells) were seeded in 100 μl per well in a flat-bottom 96-well plate. Effector cells (NK-92MI) were added in 100 μl at different effector to target cell ratios and incubated for 4 h at 37 °C. All groups were set up in triplicate. Target cells without NK cells were used for spontaneous release and target cells lysed with 1% Triton X-100 (Sigma, T8787) for maximum release. 100 μl supernatant was collected and measured using Victor X5 microplate reader (PerkinElmer) with excitation filter at 485 nm and emission filter at 535 nm. Percent lysis was calculated using the following formula:
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