Spectramax m3

Manufactured by Molecular Devices

Sourced in United States, United Kingdom, Australia, Germany, China

The SpectraMax M3 is a multimode microplate reader that can measure absorbance, fluorescence, and luminescence. It is designed to provide accurate and reliable data for a variety of assays in life science research and drug discovery applications.

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Lab products found in correlation

Methyl thiazolyl tetrazolium (mtt), by Merck Group (8 mentions) Softmax pro 6, by Molecular Devices (8 mentions) Celltiter glo, by Promega (7 mentions) Sytox green, by Thermo Fisher Scientific (7 mentions) Resazurin, by Merck Group (6 mentions) Trypsin edta solution, by Thermo Fisher Scientific (6 mentions) Amplex red hydrogen peroxide assay kit, by Thermo Fisher Scientific (5 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (5 mentions) Zetasizer nano, by Malvern Panalytical (4 mentions) Celltiter glo luminescent cell viability assay, by Promega (4 mentions) Methyl thiazolyl tetrazolium (mtt), by Duchefa Biochemie (4 mentions) Menadione, by Merck Group (4 mentions) Softmax pro, by Molecular Devices (4 mentions) Celltiter 96 aqueous one solution cell proliferation assay, by Promega (4 mentions) Dual glo luciferase assay system, by Promega (4 mentions) Luciferase assay system, by Promega (4 mentions) Cell counting kit 8 cck 8, by Dojindo Laboratories (4 mentions) Wst 1, by Roche (4 mentions) H2dcfda, by Thermo Fisher Scientific (4 mentions) Prism 6, by GraphPad (4 mentions)

910 protocols using spectramax m3

Antioxidant Activity Assessment via ABTS and DPPH

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Antioxidant activities were assessed by ABTS and DPPH assays [29 (link),30 (link)]. ABTS radicals were produced by mixing 10 mM ABTS and 10 mM potassium persulfate with a ratio of 7.4:2.6 (v/v) at 37 °C for 24 h in the dark. After dilution with PBS (absorbance in a range of 0.65 ± 0.02 at 734 nm), ABTS radical solution (150 μL) was added to the TBFEs (50 μL), and incubation was continued for 30 min at room temperature in the dark. Finally, absorbance was measured at 734 nm using a microplate reader (SpectraMax^® M3, Molecular Devices).
For the DPPH assay, DPPH solution (100 μL, absorbance in a range of 0.65 ± 0.02 at 517 nm) was added to the TBFEs (100 μL) and incubated for 30 min at room temperature. Finally, the absorbance was measured at 517 nm using a microplate reader (SpectraMax^® M3, Molecular Devices). Antioxidant activity was expressed as L-ascorbic acid equivalent (AAE g/100 g extract).

Jin H.R., Yu J, & Choi S.J. (2019). Hydrothermal Treatment Enhances Antioxidant Activity and Intestinal Absorption of Rutin in Tartary Buckwheat Flour Extracts. Foods, 9(1), 8.

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Quantification of Total Polyphenols and Flavonoids

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Total polyphenol contents were measured with the Folin–Ciocalteu method [27 (link)]. TBFEs (80 μL) were mixed with 50% Folin–Ciocalteu phenol reagent (20 μL) and incubated for 5 min in the dark. After adding 2% sodium carbonate (100 μL), the solutions were further incubated for 30 min. Finally, the absorbance was measured with a microplate reader (SpectraMax^® M3, Molecular Devices, Sunnyvale, CA, USA) at 750 nm. The contents of total polyphenols were expressed as tannic acid equivalents (TAE g/100 g extract).
Total flavonoid contents were determined using a spectrophotometric method [28 (link)]. Briefly, 5% sodium nitrite (30 μL) was added to the TBFEs (50 μL) and incubated for 5 min. After the addition of 2% aluminum chloride hexahydrate (60 μL), the mixtures were further incubated for 6 min at room temperature in the dark. Finally, 1 N sodium hydroxide (100 μL) was added and incubated for 11 min at room temperature in the dark. The absorbance was measured at 510 nm using a microplate reader (SpectraMax^® M3, Molecular Devices). Catechin was used as a standard and the results were expressed as catechin equivalent (CE g/100 g extract).

Jin H.R., Yu J, & Choi S.J. (2019). Hydrothermal Treatment Enhances Antioxidant Activity and Intestinal Absorption of Rutin in Tartary Buckwheat Flour Extracts. Foods, 9(1), 8.

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Evaluation of anti-inflammatory effects of phytosomes

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The viability of RAW264.7 cells after the 24 h incubation with VDL and VFL phytosomes (7.8–1000 µg/mL) was tested by the MTT assay. The anti-inflammatory effect of VDL and VFL phytosomes was determined from nitric oxide secretion against macrophage cells. RAW 264.7 cells (1 × 10⁴ cells/well) were incubated with LPS (50 ng/mL) in the presence or absence of VDL and VFL phytosomes (15.6–500 µg/mL) for 18 h at 5% CO₂, 37 °C. After incubation, an equal medium volume was mixed with the Griess reagent, consisting of 20 mg/mL sulfanilamide and 1 mg/mL N-(1-naphthylethylenediamine in 5% phosphoric acid at a 1:1 ratio [60 (link),61 (link)]. The absorbance of the cell supernatant was recorded at 540 nm to quantify the nitrite levels using a UV-Vis spectrophotometer microplate reader (Spectramax M3, Molecular Devices, San Jose, CA, USA) equipped with SoftMax^® Pro 7 software. The amount of nitrite was calculated from the sodium nitrite standard curve. The treated cells were then tested for cell viability using an MTT assay. The medium was replaced with 0.5 mg/mL MTT reagent (100 µL/well) and incubated for 2 h. The formazan product was measured at 550 nm using a UV-Vis spectrophotometer microplate reader (Spectramax M3, Molecular Devices, San Jose, CA, USA).

Chittasupho C., Chaobankrang K., Sarawungkad A., Samee W., Singh S., Hemsuwimon K., Okonogi S., Kheawfu K., Kiattisin K, & Chaiyana W. (2023). Antioxidant, Anti-Inflammatory and Attenuating Intracellular Reactive Oxygen Species Activities of Nicotiana tabacum var. Virginia Leaf Extract Phytosomes and Shape Memory Gel Formulation. Gels, 9(2), 78.

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Quantifying Cellular Glutathione Levels

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The glutathione levels, including both reduced (GSH) and oxidized (GSSG) glutathione, were measured with the GSH/GSSG-Glo Assay from Promega (V6611). Cells in quadruplicates were seeded at 20,000 cells/well in 96-well plates. GFPT2 knockdown and H₂O₂ treatments followed the methods described above. The glutathione levels were measured 24 h after changed medium. The luminescence signal was detected in the microplate reader (SpectraMax M3, Molecular Devices) with white and opaque 96-well plates (BRANDplates, 781965). To normalize the glutathione level, cells were counted using a crystal violet assay. In short, cells were fixed with 100% cold MeOH and stained with 0.25% crystal violet (Merck, C.I. 42555). After washing, stained cells were dissolved into 100 μl of 10% acetic acid and measured at 570 nm in the microplate reader (SpectraMax M3, Molecular Devices LLC).

Wang Q., Karvelsson S.T., Kotronoulas A., Gudjonsson T., Halldorsson S, & Rolfsson O. (2021). Glutamine-Fructose-6-Phosphate Transaminase 2 (GFPT2) Is Upregulated in Breast Epithelial–Mesenchymal Transition and Responds to Oxidative Stress. Molecular & Cellular Proteomics : MCP, 21(2), 100185.

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ABTS Radical Scavenging Assay for B. hispida Peel

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The potential of B. hispida peel extracts was assessed in terms of scavenging activity using the ABTS assay following the method of Poomanee [21 (link)] with modifications. The ABTS reagent was prepared by mixing an ABTS solution in DI water (7 mM) with potassium persulfate solution in DI water (3 mM) in a ratio of 1:0.5 and incubated in a dark area for 18 h. The measurement was performed using a microplate reader (SpectraMax M3, Molecular Devices) at 734 nm. Before testing, the prepared ABTS reagent was diluted to provide an absorbance of 0.7 and was then used as a tested ABTS reagent. Altogether, 20 µL of the extract solution (0.0 to 1.0 mg/mL) and 180 µL of tested ABTS reagent were mixed and incubated in the dark at ambient temperature for 30 min. The absorbance of the extract–reagent mixture was measured at 520 nm using a microplate reader (SpectraMax M3, Molecular Devices). The % inhibition against ABTS radicals (% inhibition) was estimated using Equation (2) described above.

Phumat P., Chaichit S., Potprommanee S., Preedalikit W., Sainakham M., Poomanee W., Chaiyana W, & Kiattisin K. (2023). Influence of Benincasa hispida Peel Extracts on Antioxidant and Anti-Aging Activities, including Molecular Docking Simulation. Foods, 12(19), 3555.

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Antioxidant Activity of Quercetin and Rutin

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Antioxidant activity of quercetin or rutin (312.5 μM) in the presence of ZnO NPs (100 μg/mL) was evaluated by radical ABTS and DPPH scavenging assays. The ABTS radicals were generated by mixing 10 mM ABTS and 10 mM K₂SO₄ with a ratio of 7.4:2.6 (v/v) at 37 °C for 24 h in the dark. After dilution with PBS to appropriate concentrations, 150 μL of ABTS radical solution was added to 50 μL of the samples, and the solutions were incubated at room temperature in the dark. After 30 min, absorbance was measured at 734 nm using a microplate reader (SpectraMax^® M3, Molecular Devices). Antioxidant activity was expressed as L-ascorbic acid equivalent (AAE mg/100 mL sample).
For the DPPH assay, 100 μL of DPPH solution was added to 100 μL of the samples, and the solutions were incubated at room temperature. After 30 min, the absorbance was measured at 517 nm using a microplate reader (SpectraMax^® M3, Molecular Devices). Antioxidant activity was expressed as L-ascorbic acid equivalent (AAE mg/100 mL sample).

Kim S.B., Yoo N.K, & Choi S.J. (2022). Interactions between ZnO Nanoparticles and Polyphenols Affect Biological Responses. Nanomaterials, 12(19), 3337.

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Polystyrene Bead Uptake Quantification

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Twenty five μL of effluent from each sampling time point was pipetted in duplicate on a 96-well clear bottom plate. The positive control was identified as 25 μL of stock solution of 20 nm rhodamine-labeled polystyrene beads and the negative control as PSS only. All samples were diluted by adding 100 μL of PSS into each well. Fluorescence was measured by a spectrophotometer at 546/575 nm (excitation/emission) using a SpectraMax M3 fluorescent microplate reader (Molecular Devices, Sunnyvale, CA). Data were collected using SoftMax Pro 6.3 software.
To confirm that the rhodamine tag remained attached to the polystyrene beads throughout the perfusion, maternal and fetal effluents were pooled together for 4 representative experiments. The samples were centrifuged at 100,000 x g for 1 h in an ultracentrifuge (Beckman Coulter Max-XP tabletop Ultracentrifuge) to pellet polystyrene ENM. Twenty five μL of supernatant was removed from each sample and placed in a 96-well clear bottom plate and read at 546/575 nm (excitation/emission) using a SpectraMax M3 fluorescent microplate reader (Molecular Devices, Sunnyvale, CA). Data were collected using SoftMax Pro 6.3 software.

Fournier S.B., D’Errico J.N., Adler D.S., Kollontzi S., Goedken M.J., Fabris L., Yurkow E.J, & Stapleton P.A. (2020). Nanopolystyrene translocation and fetal deposition after acute lung exposure during late-stage pregnancy. Particle and Fibre Toxicology, 17, 55.

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Quantification of H2O2 Release Across Tissues

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H₂O₂ release was quantified using Amplex^® red (Molecular Probes, USA) in the presence of horseradish peroxidase (HRP). Isolated-mitochondria: The H₂O₂ release in isolated mitochondria was determined as described previously
³³⁾. Aorta: Two 3 mm rings of the descending aorta were placed in a well of a 96-well plate with Amplex^® red (10 µM) and HRP (0.2 U/mL) in PBS buffer containing 11.1 mM glucose, pH 7.4, for 1 h. Then, the aortic rings were removed, and the fluorescence signal was measured in the buffer at the excitation/emission wavelengths of 530/590 nm (SpectraMax M3, Molecular Devices, USA)
³⁴⁾. Peritoneal macrophages: Experiments were done in a 96-well plate in 100 µl of PBS buffer containing 11.1 mM glucose, pH 7.4, in the presence of Amplex^® red (25 µM) and HRP (0.2 U/mL) in the dark at 37℃ for 40 min; fluorescence (excitation: 530 nm, emission: 590 nm) was measured every 10 min (SpectraMax M3- Molecular Devices, USA). Catalase (500 U/mL) was added at least in one well per group as a background control to account for nonspecific signal. Phorbol 12-myristate 13-acetate (PMA) (100 nM) was used to activate the macrophages during the assay.

Dorighello G.G., Rovani J.C., Paim B.A., Rentz T., Assis L.H., Vercesi A.E, & Oliveira H.C. (2021). Mild Mitochondrial Uncoupling Decreases Experimental Atherosclerosis, A Proof of Concept. Journal of Atherosclerosis and Thrombosis, 29(6), 825-838.

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Colorimetric Assay for Parasite Hsp70 ATPase

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The basal ATPase activities of the parasite Hsp70s was assessed by a colourimetric-based method, as previously described [27 (link)]. Briefly, 0.4 µM of recombinant proteins (PfHsp70-1/PfHsp70-z) was incubated in buffer HKMD (10 mM HEPES-KOH pH 7.5, 100 mM KCl, 2 mM MgCl2, 0.5 mM DTT) at 37 °C. The reaction was started by the addition of saturating 5 mM ATP and samples were collected after every 30 min for 4 h. The released inorganic phosphate was detected by adding 1.25% ammonium molybdate and 9% ascorbic acid and monitored using a M3 SpectraMax (Molecular Devices, Sunnyvale, CA, USA) spectrophotometer at 660 nm. To determine the effect of EGCG on the ATPase activity of parasite Hsp70s, the assay was repeated in the presence of varying amounts (0–5 µM) of EGCG. As controls, boiled Hsp70 protein was used to account for spontaneous ATP hydrolysis, and BSA was used as a non-chaperone control.

Zininga T., Ramatsui L., Makhado P.B., Makumire S., Achilinou I., Hoppe H., Dirr H, & Shonhai A. (2017). (−)-Epigallocatechin-3-Gallate Inhibits the Chaperone Activity of Plasmodium falciparum Hsp70 Chaperones and Abrogates Their Association with Functional Partners. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(12), 2139.

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Cell Viability Assay Using CCK-8

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For measuring cell viability we performed the cell proliferation assay (CCK-8) as per the manufacturer (Sigma-Aldrich, St. Louis, MO) recommendations. 100ul of cell suspension containing 5000 cells was seeded in a 96-well plate. After 24 h pre-incubation at 37°C in 5% CO₂, the cells were incubated in room or hyperoxia (95% O₂, 5% CO₂). 10 μl CCK-8 solution was added to each well in the last 1 hour of incubation. Absorbance at 450 nm was measured at 0, 8, 24 and 48 hr after hyperoxia incubation using a microplate reader (M3 SpectraMax, Molecular Devices Corporation, Sunnyvale, CA).

Zhang Y, & Lingappan K. (2017). Differential Sex-Specific Effects of Oxygen Toxicity in Human Umbilical Vein Endothelial Cells. Biochemical and biophysical research communications, 486(2), 431-437.

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