Cells (2 × 104 per well) for measuring proliferation and cells (5 × 104 per well) for testing compound cytotoxicity were seeded into 24-well plates. After overnight incubation, cells were treated with different concentrations of bestatin. Cells for measuring cytotoxicity were incubated for 24 or 48 h and cells for measuring proliferation were incubated for 24, 48, or 72 h. After washing 3 times with phosphate buffered saline (PBS), cells from each different time point were fixed with methanol for 10 min and stained with 0.2% (w/v) crystal violet in 2% (v/v) ethanol for 10 min. Then, 0.5% (w/v) sodium dodecyl sulfate in 50% (v/v) ethanol was added to each well. Absorbance was measured at an optical wavelength of 540 nm using the Thermo Multiskan plate-reader (Thermo Fisher Scientific, Waltham, MA, USA).
Thermo multiskan plate reader
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, China
The Thermo Multiskan plate-reader is a laboratory instrument used for performing photometric measurements on microplates. It can quantify the absorbance of samples within the microplate wells, allowing for various analytical applications such as enzyme-linked immunosorbent assays (ELISA), cell-based assays, and colorimetric biochemical assays.
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Lab products found in correlation
Celltiter 96 aqueous one solution, by Promega (4 mentions)
Trypsin, by Merck Group (3 mentions)
Mtt solution, by Solarbio (2 mentions)
Foetal calf serum, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Nunclon microplates, by Thermo Fisher Scientific (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Vero cells, by Merck Group (1 mentions)
Multisizer 4e coulter counter, by Beckman Coulter (1 mentions)
Penicillin, by Merck Group (1 mentions)
18 protocols using thermo multiskan plate reader
1
Bestatin's Effects on Cell Proliferation and Cytotoxicity
2
MTT Assay for Cell Viability and IC50 Determination
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HCC cells were seeded in 96-well culture plates at a density of 2000 cells/well. After a certain period of incubation, 20 μL of 5 mg/mL MTT (Biosharp, Hefei) was added to each well and incubated at 37°C for 2 h. Formazan crystals were dissolved in 100 μL of DMSO. Absorbance was measured at 490 nm using a Thermo Multiskan plate reader (Thermo Fisher Scientific, China). When measuring the 50% inhibitory concentration (IC50) value, 5000 cells were seeded in each well; epirubicin (Hisun, Zhejiang) was added after overnight incubation; ten different measurement concentrations of 0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, and 25.6 mg/mL were set; and the absorbance was measured after culturing for 48 h. Each independent experiment was repeated at least three times.
3
Melanocyte Cell Cytotoxicity Assay
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Melanocyte cells (1×104 cells/well) were seeded into 96-well plates in 100 mL of medium. After 24 h of culture, the appropriate concentration of each compound was added to each well. After incubation for another 24 h or 48 h, 20 μL of the CellTiter 96 Aqueous One Solution (Promega Corporation, Fitchburg, WI) were added to each well and cells were incubated for an additional 1 h. Absorbance was measured at 492 nm using the Thermo Multiskan plate-reader (Thermo Fisher Scientific, Waltham, MA).
4
Cell Proliferation Assay with Crystal Violet
5
Quantification of Bmi-1 and MMP9 Proteins
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The Bmi-1 kit used for ELISA was purchased from Biomatik (EKC35069) and the MMP9 kit purchased from Thermo Fisher Scientific, Inc. (KHC3061). Briefly, 50 µl of standard and sample was added to the plate, cultured for 2 h at room temperature, and washed 4 times using the wash buffer from the kit. Then 100 µl of antibody was added and incubated for 1 h in room temperature, followed by washing 4 times. HRP conjugate was added and cultured for 30 min at room temperature and the wells washed 4 times and treated with chromogenic substrate. Finally, the results were defined by absorbance at 450 and 55 nm using a Thermo Multiskan plate reader (Thermo Fisher Scientific, Inc.); each experiment was replicated 3 times.
6
NF-κB Transcriptional Activity Assay
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The assay was performed using a Dual Luciferase Assay kit (Promega Corporation, Madison, WI, USA). First the promoter region of NF-κB was cloned to a luciferase vector, MG-63 cells were seeded onto a 24-well plate, 30,000 cells in each well, and then the cells were transfected with luciferase and Renilla. After 24 h, luciferase and Renilla activity was detected by a Thermo Multiskan plate reader (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Activity of NF-κB was defined as the ratio of firefly luciferase activity compared with corresponding Renilla luciferase activity. Each experiment was repeated by 3 times.
7
Anwulignan Cytotoxicity Assay in Lung Cancer Cells
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A549, H1299, H1650 and H1975 cells (2 × 103 cells per well) were seeded for 24 hours in 96‐well plates. Various concentrations of Anwulignan were then added to each well, and cells were incubated for 48 hours. Twenty μl of MTT solution (Solarbio, Beijing, China) was added to each well; the cells were then incubated for 2 hours at 37°C in a 5% CO2 incubator. Next, the cell culture medium was discarded and replaced with 200 µl of DMSO. Formazan crystals were dissolved by gentle agitation. Finally, cell growth was analysed at 570 nm absorbance using the Thermo Multiskan plate‐reader (Thermo Fisher Scientific, Waltham, MA, USA).
8
Cytotoxicity Assessment of Ricin Toxin
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Vero cells (ECACC 84113001) were obtained from the European Collection of Animal Cell Cultures (ECACC) (Public Health England, Salisbury, UK). Cells were maintained in culture medium consisting of DMEM (Sigma Aldrich, Poole, UK) with 10% (v/v) foetal calf serum (Sigma Aldrich, Poole, UK), 1% penicillin/streptomycin solution (containing 100 units·mL−1 penicillin and 0.01 mg·mL−1 streptomycin), and 1% (w/v) l -glutamine (Sigma Aldrich, Poole, UK) 2 mM. Vero cells were grown in 150 cm2 flasks in a humidified atmosphere of 5% CO2 in air at 37 °C and removed from the flask surface using incubation with trypsin (0.05% w/v) (Sigma Aldrich, Poole, UK) containing EDTA (0.03% w/v) (Sigma Aldrich, Poole, UK) on achieving 70–90% confluency for seeding into 96 well test plates (cell density of 5 × 103 cells per well). The cells were allowed to adhere to the culture plates for 24 h before use.
For toxicity assessment, ricin toxin was diluted to 100 ng·mL−1 in culture medium and filtered using a 0.2 µm sterile filter before further dilution in culture medium and addition to the assay plate in triplicate. The plates were then incubated for 48 h prior to the addition of 10 µL of Roche Cell Proliferation Reagent WST-1 (Sigma Aldrich, Poole,UK). After 3 h the absorbance was read on a Thermo Multiskan plate reader (Thermo Fisher Scientific, Loughborough, UK) at 450 nm to assess cell viability.
For toxicity assessment, ricin toxin was diluted to 100 ng·mL−1 in culture medium and filtered using a 0.2 µm sterile filter before further dilution in culture medium and addition to the assay plate in triplicate. The plates were then incubated for 48 h prior to the addition of 10 µL of Roche Cell Proliferation Reagent WST-1 (Sigma Aldrich, Poole,UK). After 3 h the absorbance was read on a Thermo Multiskan plate reader (Thermo Fisher Scientific, Loughborough, UK) at 450 nm to assess cell viability.
9
Algal Toxicity of Pesticides TEB and 124T
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The toxicity of TEB and 124T to phytoplankton was examined in inhibition tests with the unicellular green microalgae Raphidocelis subcapitata (formerly Selenastrum capricornutum and Pseudokirchneriella subcapitata). The endpoint was inhibition of growth measured after 72 h of incubation as described in ISO 8692 (2012 ). R. subcapitata (MicroBioTests Inc.) was cultivated in alga test medium at 22 ± 2 °C and continuous illumination at 6500 lx (ISO 8692 , 2012 ). Two-fold dilutions of TEB or 124T were prepared in 96-well clear Nunclon microplates (Thermo Scientific) by serially diluting 150 µL of an aqueous solution with the chemical in 150 µL algal test medium. After transfer of the chemicals, 150 µL of diluted R. subcapitata culture (1:50) was added to each well resulting in a final liquid volume of 300 µL in each well. Plates were incubated for 72 h at 22 ± 2 °C on a shaker at 70 rpm with continuous illumination (6500 lx). Growth was measured after 0, 24 h, 48 h, and 72 h as absorbance at 450 nm using a Thermo Multiskan Plate Reader (Thermo Scientific). The bioassay with R. subcapitata included eight replicates of blanks (medium only), controls (no test chemical), and 10 nominal concentrations of TEB and 124T. The toxicity of TEB to R. subcapitata was examined before and after exposure of aqueous solutions to different VUV/UVC irradiation regimes.
10
Cytotoxicity Assay for ADA-07 Treatment
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