Prestoblue assay

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom, Germany

PrestoBlue assay is a fluorometric cell viability reagent. It is used to measure the reducing capacity of cells, which is an indicator of cell health and proliferation.

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PrestoBlue (489 citations) PrestoBlue cell viability assay (70 citations) Prestoblue assay kit (8 citations) Prestoblue cell viability assay reagent (5 citations) PrestoBlue™ Viability Reagent reduction assay (3 citations) PrestoBlue™ HS Cell Viability assay (3 citations) PrestoBlue Cell Viability Assay kit (3 citations) PrestoBlue® (PB) assay (3 citations) PrestoBlue resazurin assay (2 citations) PrestoBlue metabolic activity assay (2 citations)

130 protocols using prestoblue assay

Biofunctional Polymer Synthesis and Evaluation

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D,L-lactide was provided by Corbion (Gorinchem, The Netherlands). Tin (II) 2-ethylhexanoate (Sn(Oct)₂, 95%), pentaerythritol, diethylene glycol (DEG), (3-isocyanatopropyl) triethoxysilane (IPTES), dichloromethane, heptane, toluene, tetrahydrofuran, hydrochloric acid (37%) and hydrochloric acid (37%) (1 M in methanol), dichloromethane (DCM), trifluoroethanol, trifluoroacetic acid and phosphate buffer solution (PBS) were purchased from Sigma-Aldrich (St Quentin Fallavier, France).
CellTiter Glo assay was provided by Promega G7571 (Charbonnières-les-Bains, France). PrestoBlue^® assay (A13262) and Clariostar plate reader (A13626) were acquired from Invitrogen (Illkirch, France). Negative RM-C and positive RM-A were supplied by Hatano Research Intitue, Food and Drug Safety Center, Hadano, Japan).

Belabbes K., Pinese C., Leon-Valdivieso C.Y., Bethry A, & Garric X. (2022). Creation of a Stable Nanofibrillar Scaffold Composed of Star-Shaped PLA Network Using Sol-Gel Process during Electrospinning. Molecules, 27(13), 4154.

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Cell Viability Assay with PrestoBlue

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HREC were plated 20,000/well in a 96-well plate, and cell viability was determined by PrestoBlue^® assay (Invitrogen, Waltham, MA, USA), following the manufacturer’s instructions and as previously reported [31 (link)]. After treatments, cells were loaded for 30 min with PrestoBlue^® reagent prior to assay readout. Fluorescence was measured by the Synergy HT multidetection microplate reader (BioTek, Winooski, VT, USA), with excitation and emission wavelengths of 530 and 590 nm, respectively. The results are expressed as a percentage of the fluorescence of the samples in comparison to control (100%).

Manai F, & Amadio M. (2022). Dimethyl Fumarate Triggers the Antioxidant Defense System in Human Retinal Endothelial Cells through Nrf2 Activation. Antioxidants, 11(10), 1924.

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Cytotoxicity Evaluation of Entry Inhibitors

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Huh7.5.1 cells and PHH were incubated with compounds for 48 h and/or 5 days.22 (link)
23 (link) Cytotoxic effects were analysed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay13 (link)
22 (link) or PrestoBlue assay (Invitrogen) with flavopiridol or anti-Fas antibody as positive controls.22 (link) The 50% cytotoxic concentrations (CC₅₀) of entry inhibitors were calculated by regression analysis.

Xiao F., Fofana I., Thumann C., Mailly L., Alles R., Robinet E., Meyer N., Schaeffer M., Habersetzer F., Doffoël M., Leyssen P., Neyts J., Zeisel M.B, & Baumert T.F. (2014). Synergy of entry inhibitors with direct-acting antivirals uncovers novel combinations for prevention and treatment of hepatitis C. Gut, 64(3), 483-494.

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Evaluating hDPSCs Viability in 3D Scaffolds

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Human dental pulp stem cells (hDPSCs) were purchased from Lonza (PT-5025, Lonza, Basel, Switzerland) in a commercially-available human dental pulp stem cell bullet kit (PT-3005, Lonza, Basel, Switzerland). New culture medium was replaced twice a day in a 37 °C humidified atmosphere with 5% CO₂. The cells started the experiment when the culture reached passage 3–6; the 3D scaffolds were pretreated with 75% ethanol for 1h, then subjected to ultraviolet light exposure for 30 min before the cell study. After the various duration of cultures, the PrestoBlue assay (Invitrogen, Carlsbad, CA, USA) was utilized, following the manufacturer’s manual instructions to experiment. Internal mitochondrial activity was investigated according to the color intensity of the reagent. The viability value was measured by a multi-well spectrophotometer (Infinite Pro M200, Tecan, Männedorf, Switzerland) at 570 nm with a reference wavelength of 600 nm. The only hDPSCs seeding was the control group.

Chen C.Y., Shie M.Y., Lee A.K., Chou Y.T., Chiang C, & Lin C.P. (2021). 3D-Printed Ginsenoside Rb1-Loaded Mesoporous Calcium Silicate/Calcium Sulfate Scaffolds for Inflammation Inhibition and Bone Regeneration. Biomedicines, 9(8), 907.

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Cell Adhesion Kinetics on Biomaterials

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The suspended cells were kept at a density of 1.5 × 10⁴/specimen that were directly seeded over each sample in different DMEMs, after which the cell cultures were incubated at 37 °C in a 5% CO₂ atmosphere. After being cultured for various pre-determined lengths of time (3, 6, 12 h, and 1, 3, 7 days), cell adhesion ability was evaluated using the PrestoBlue^® assay (Invitrogen, Grand Island, NY, USA). To describe the process briefly, each specimen was filled at a 1:9 ratio of PrestoBlue^® in fresh DMEM and incubated at 37 °C for 30 min. The solution in each well was then transferred to a new 96-well plate and read using a multiwell spectrophotometer (Hitachi, Tokyo, Japan) at 570 nm with a reference wavelength of 600 nm. The results were obtained in triplicate from three separate experiments for each test. Cells cultured on tissue culture plates without cement were used as a control (Ctl).

Lai W.Y., Chen Y.W., Kao C.T., Hsu T.T., Huang T.H, & Shie M.Y. (2015). Human Dental Pulp Cells Responses to Apatite Precipitation from Dicalcium Silicates. Materials, 8(7), 4491-4504.

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Measuring Cell Viability in Hypoxia

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Cell viability was measured using Prestoblue assay (Invitrogen) and performed according to the manufacturer's protocol. Briefly, PC-3 cells were seeded at a density of 5000 cells/well. Cells were pre-treated with inhibitors for 30 min before incubation at 1% O₂ or 21% O₂ for 24 h. The absorbance was recorded at 570 nm after 30 min incubation of cells with Presto Blue reagent. The cell viability was expressed as a percentage relative to untreated controls.

Ivanova I.G., Park C.V., Yemm A.I, & Kenneth N.S. (2018). PERK/eIF2α signaling inhibits HIF-induced gene expression during the unfolded protein response via YB1-dependent regulation of HIF1α translation. Nucleic Acids Research, 46(8), 3878-3890.

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Evaluating EPC Viability with mRNA Treatment

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The influence of mRNA treatment on the viability of EPCs was assessed using PrestoBlue assay (Invitrogen, Carlsbad, CA, USA). Therefore, 1 × 10⁵ EPCs were transfected with 1.6 μg ANG-1, 0.8 μg VEGF-A, or 0.5 μg SDF-1α mRNA or with a triple-mRNA cocktail containing 1.6 μg ANG-1, 0.8 μg VEGF-A, and 0.5 μg SDF-1α mRNA for 4 hr at 37°C. Afterward, transfection complexes were discarded and 1 mL cell culture medium was added per well of a 12-well plate. After 24 hr, 110 μL PrestoBlue cell viability reagent was added per well and incubated for 1.5 hr at 37°C. Fluorescence intensity of 50 μL supernatant was measured in triplicates at an excitation wavelength of 530 nm and an emission wavelength of 600 nm, using a multimode microplate reader (Mithras LB 940, Berthold Technologies).

Steinle H., Golombek S., Behring A., Schlensak C., Wendel H.P, & Avci-Adali M. (2018). Improving the Angiogenic Potential of EPCs via Engineering with Synthetic Modified mRNAs. Molecular Therapy. Nucleic Acids, 13, 387-398.

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Optimizing Tri-Culture Lung Cell Growth

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This is the first study that incorporates these three different kinds of alveolar lung cells together. After some optimization, a 1:1 v/v combination of RPMI 1640 and DMEM/F12 was found to support the co-culture of A549, EAHy926 and MRC5. Hence, the cell proliferation profile in the proposed co-culture medium was evaluated against their respective recommended medium (benchmark). Before the bioprinting process, the three different types of human pulmonary cell lines were cultured individually in the co-culture medium (1:1 v/v of RPMI-1640 to DMEM/F12 culture medium – Gibco™ Thermo Fisher Scientific) and their respective culture medium (control medium), to assess the suitability of the co-culture medium to support the growth of the different cell types. The PrestoBlue^® assay (Invitrogen™ A13262, Thermo Fisher Scientific) was used to measure cell proliferation based on the normalized relative fluorescence units over a period of 7 days; with the cell morphologies and proliferation rates serving as indicators for determining the suitability of the co-culture medium.

Ng W.L., Ayi T.C., Liu Y.C., Sing S.L., Yeong W.Y, & Tan B.H. (2021). Fabrication and Characterization of 3D Bioprinted Triple-layered Human Alveolar Lung Models. International Journal of Bioprinting, 7(2), 332.

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Evaluating Metabolic Activity in MC3T3-E1 Cells

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To assess cell metabolic activity, MC3T3-E1 pre-osteoblasts were seeded on OCP-coated titanium discs without/with κ-carrageenan at 1×10⁵ cells/disc in 48-well plates (Greiner), and cultured up to 7 days. The medium was removed, cells were washed with PBS, and α-MEM with 10% FBS and antibiotics was added. The PrestoBlue™ Assay (Invitrogen) was used to evaluate cell metabolic activity according to the manufacturer’s instructions. In short, PrestoBlue™ reagent was added to the cells (10%, vol/vol), followed by 30 min incubation in a 5% CO₂ in air incubator with a humidified atmosphere at 37°C. The medium was harvested (100 μl/well) and transferred into a 96-wells black microplate (Greiner). Fluorescence intensity was determined at a wavelength of 560 nm (excitation) and 590 nm (emission), and quantified using a Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific). Prestoblue™ fluorescence was linearly associated with DNA content (data not shown).

Cao W., Jin J., Wu G., Bravenboer N., Helder M.N., Schulten E.A., Bacabac R.G., Pathak J.L, & Klein-Nulend J. (2022). Kappa-carrageenan-Functionalization of octacalcium phosphate-coated titanium Discs enhances pre-osteoblast behavior and osteogenic differentiation. Frontiers in Bioengineering and Biotechnology, 10, 1011853.

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Evaluating hMSC Viability in GelMA Hydrogels

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To evaluate cell survival, 1 × 10⁶ hMSCs were encapsulated in 1 mL of 10% (w/v) GelMA hydrogels. Cell viability was evaluated by staining hMSCs encapsulated in GelMA hydrogel using Live/Dead assay (Invitrogen, USA) according to the manufacturer’s instructions after 1, 3, 6, and 12 days of culture. Stained cells were visualized and microphotographed using fluorescence microscopy (Zeiss, Axio Observer A.1). The metabolic activity of encapsulated cells was assessed using PrestoBlue assay (Invitrogen, USA) according to the manufacturer’s protocol and measured using a BioTek Synergy 2 plate reader.

Farzin A., Hassan S., Teixeira L.S., Gurian M., Crispim J.F., Manhas V., Carlier A., Bae H., Geris L., Noshadi I., Shin S.R, & Leijten J. (2021). Self-Oxygenation of Tissues Orchestrates Full-Thickness Vascularization of Living Implants. Advanced functional materials, 31(42), 2100850.

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