The mitochondrial activity assessed by the PrestoBlue™ solution (#A13261, Thermo Fisher Scientific) was used to evaluate cell viability. A total of 100 µL of the reagent diluted 1:10 in the cultivation medium was added to one part of the bioengineered intestinal tubule and incubated at 37 °C 5% CO2 for 1 h, protected from light. Fluorescence resulting from the reduction of PrestoBlue™ was measured on GloMax® Discover (Promega) set at 520 nm excitation and 580–640 nm emission wavelengths. Values were corrected for bioengineered intestinal tubule length, and measured values were calculated relative to the medium-only control.
Prestoblue solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
PrestoBlue solution is a cell viability reagent. It is a resazurin-based solution that can be used to measure the metabolic activity of cells in a sample. The solution changes color based on the reduction of resazurin to resorufin, which is an indicator of cell viability.
Automatically generated - may contain errors
Lab products found in correlation
Glomax discover, by Promega (1 mentions)
Victor x4 2030 multilabel reader, by PerkinElmer (1 mentions)
Picogreen dsdna reagent, by Thermo Fisher Scientific (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Streptomycin sulfate, by Merck Group (1 mentions)
Black 96 well plate, by Corning (1 mentions)
24 well culture plate, by Corning (1 mentions)
Epoch microplate spectrophotometer, by Agilent Technologies (1 mentions)
Spectramax m2e microplate reader, by Molecular Devices (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Victor plate reader, by PerkinElmer (1 mentions)
9 protocols using prestoblue solution
1
Cell Viability Assay for Intestinal Tubules
2
Assessing Adherent Cell Viability
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ADHLSCs were seeded at 5,000 cells/cm2 on CellBIND® dishes
or on a thermosensitive polymer, using the UpCellTM technology. Upon
reaching 70% to 80% confluence, the prestoblue assay was performed by removing
the culture medium and replacing it with complete medium containing a 1/10
dilution of the prestoBlue solution (ThermoFisher Scientific). After 24 h, the
resulting fluorescence was measured using a VICTOR X4 2030 Multilabel Reader
(Perkin Elmer, Zaventem, Belgium).
or on a thermosensitive polymer, using the UpCellTM technology. Upon
reaching 70% to 80% confluence, the prestoblue assay was performed by removing
the culture medium and replacing it with complete medium containing a 1/10
dilution of the prestoBlue solution (ThermoFisher Scientific). After 24 h, the
resulting fluorescence was measured using a VICTOR X4 2030 Multilabel Reader
(Perkin Elmer, Zaventem, Belgium).
3
Magnetic Levitation and Hanging Drop Cytometry
4
Cell Viability and Proliferation Assay
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The culture medium was removed after 3 and 7 days of cell culture and the samples were transferred to new 12-well plates; subsequently, 10% PrestoBlue solution was added (5 mg/mL in DMEM; Thermo Scientific, Oxford, UK) and the multi-well plates were incubated at 37 °C for 2 h. After the supernatant removal, the solution was transferred in 96-well plates (0.2 mL) and quantified with a spectrophotometer working at 560 nm; a Filter-based FLUOstar® Omega multi-mode reader (FLUOstar® Omega, BMG Labtech, Ortenberg, Germany) was used. PicoGreen® dsDNA reagent purchased from Invitrogen (Carlsbad, CA, USA) was employed to calculate the number of the cells for each sample to make a correct normalisation of the fluorescence values. The scaffolds were carefully washed with PBS after each culturing period, incubated for 3 h at 37 °C for 3 h and then frozen at −80 °C overnight in ultra-pure water (1 mL) in order to ensure cell lysis. The assay was carried on according to the protocol of the manufacturer. Fluorescence was measured at an excitation and emission wavelength of 485 and 528 nm, respectively.
5
Microbial Growth Inhibition Assay
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The minimal inhibitory concentration (MIC) and half-maximal inhibitory concentration (IC50) values for E. amylovora TS3128 were determined by the two-fold serial dilution assay as described previously (Espinel-Ingroff et al., 2005 (link)). Briefly, a bacterial suspension (2 × 105 cfu/ml of MGY) of E. amylovora TS3128 was added to the wells of a 96-well microtiter plate. The culture filtrates were added to the well at an initial concentration of 20% and then serially two-fold diluted. The purified compounds dissolved in dimethyl sulfoxide (DMSO) were added and then serially two-fold diluted to reach the final concentrations ranging from 0.1 to 200 μg/ml; the final concentration of DMSO did not exceed 1%. The antibiotic streptomycin sulfate (Sigma-Aldrich, St. Louis, MO, USA) and an MGY medium containing 1% DMSO were used as positive and negative controls, respectively. The microtiter plates were incubated for 24 h at 28°C, and the MIC values were determined by visual inspection of complete growth inhibition. IC50 values was calculated as follows: [1 – (OD600 of treatment/OD600 of control) × 100]. The inhibitory effects on the bacterial growth were also visualized by Prestoblue solution (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. The assay was performed two times with three replicates at all concentrations investigated.
6
Cell Viability Assay via PrestoBlue
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Cells were seeded at varying concentrations of 1.5, 2.0, 2.5, 3.0, 3.5×104cells/ml in a 24-well culture plate (Corning, USA) to achieve a range of cell confluencies between 20% and 90% over a period of 4 days. Cell viability was assessed each day using a 10% PrestoBlue solution (Thermo Fisher Scientific, USA). This was done by adding the PrestoBlue solution to wells with cells that have reached the desired confluencies and subsequently incubating for 1h. 100μl of media was then transferred to a black 96-well plate (Corning, USA) before fluorescence intensity measurements in a multiplate reader (Tecan Infinite M200 Pro, Thermo Fisher Scientific, USA). Measurements were made at 560nm excitation with emission collection at 590nm. This was conducted over three biological repeats, with at least three sets of data per reported confluency.
7
PrestoBlue Viability Assay Protocol
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PrestoBlue® assay was performed according to the manufacturer's instructions. Each well contained the cells cultured medium removed. PrestoBlue® solution (Invitrogen) was added to each well, and the plates were incubated at 37℃ with 5% CO2 for a specified duration. After incubation, the absorbance was measured using an Epoch microplate spectrophotometer (BioTek Instruments, Winooski, VT, USA) at 570 nm.
8
Measuring IL-4 and NF-κB in LPS-challenged MSCs
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IL-4 expression by unaltered MSCs and NF-κB sensing IL-4 MSCs with LPS challenge was measured. Cells were seeded in 24-well plates at a concentration of 1 × 104/well, with supplemented culture medium until 80% confluency. Fresh medium was then changed, and cells were cultured with 1 μg/ml LPS (Sigma-Aldrich, St. Louis, MO) or left untreated for 24 h. Supernatant was collected for Enzyme-Linked Immunosorbent Assay (ELISA kit for mouse IL-4, R&D system, Minneapolis, MN, United States). The optical densities were determined using SpectraMax M2e Microplate Readers (Molecular Devices, San Jose, CA, United States) set at 450 nm with wavelength correction set to 540 nm. Cell viability was assessed with PrestoBlue assay (Xu et al., 2015 (link)). After collection of supernatant, 1 ml of PrestoBlue solution (10% in medium without serum and phenol red, Invitrogen, Carlsbad, CA, United States) was added to each well, and the plates were incubated at 37°C for 30 min. After incubation, 100 μL of the PrestoBlue solution from each well was transferred to a new well in 96-well plate, and the fluorescence of the test reagent was measured with the excitation/emission wavelengths set at 560/590 nm.
9
Optimizing ECM Production Assay
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Three different concentrations of each hit compound were tested for their capacity to enhance ECM production. The experimental protocol was the same as the primary screen, with the exception that each compound and concentration was tested in triplicate. A PrestoBlue assay was performed to assess the effect of the compound on metabolic activity, representing cell viability. Briefly, BM containing 10% (v/v) PrestoBlue solution (Invitrogen) was added and incubated at 37°C for 1h. Then, fluorescence was measured at 590 nm on a Victor plate reader (PerkinElmer). The concentration that induced the highest secretion of cECM without affecting the metabolic activity was selected for subsequent experiments.
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