Spectramax m3 microplate reader

Manufactured by Molecular Devices

Sourced in United States

The SpectraMax M3 is a microplate reader designed for absorbance, fluorescence, and luminescence detection. It features an adjustable wavelength range and can measure multiple detection modes simultaneously. The reader is capable of handling a variety of microplate formats.

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Lab products found in correlation

Celltiter glo luminescent cell viability assay, by Promega (4 mentions) Amplex red reagent, by Thermo Fisher Scientific (3 mentions) Softmax pro software, by Molecular Devices (3 mentions) Methyl thiazolyl tetrazolium (mtt), by Merck Group (3 mentions) Dimethyl sulfoxide (dmso), by Avantor (2 mentions) Pyrogent 5000 kit, by Lonza (2 mentions) Softmax pro, by Molecular Devices (2 mentions) Pezx mt06, by GeneCopoeia (2 mentions) Dual luciferase reporter assay kit, by Promega (2 mentions) Spectramax pro software, by Molecular Devices (2 mentions) Celltiter glo reagent, by Promega (2 mentions) Fitc dextran, by Merck Group (2 mentions) Cell titer glo 2.0 luminescence assay, by Promega (2 mentions) Luciferase assay system, by Promega (2 mentions) Dual luciferase reporter assay system, by Promega (2 mentions) Biolux gaussia luciferase assay kit, by New England Biolabs (2 mentions) 3 3 5 5 tetramethylbenzidine substrate solution, by Thermo Fisher Scientific (2 mentions) Maxisorp plate, by Thermo Fisher Scientific (2 mentions) Human albumin, by Merck Group (2 mentions) Cell counting kit 8 cck 8, by Beyotime (2 mentions)

138 protocols using spectramax m3 microplate reader

Measuring Cellular Glutathione and Succinate

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Glutathione content was measured under normoxic conditions and after exposure to 30 min and 6 h hypoxia using the GSH-Glo Assay Kit (Promega, Madison, WI, USA) according to manufacturer instructions. Luminescence was measured using a SpectraMax M3 Microplate Reader (Molecular Devices, San Jose, CA, USA). Changes in GSH levels were also measured in hypoxic live cells using ThiolTracker Violet (Invitrogen, catalog # T10096). Cells were loaded with 20 µM ThiolTracker Violet glutathione detection reagent and 100 nM SYTO 16 Green Fluorescent Nucleic Acid Stain (Invitrogen, catalog # S7578) and imaged within the hypoxic environment using an LS620 microscope (Etaluma, Carlsbad, CA, USA) fitted with a 20 × objective at 0, 30 min, and 6 h at 1% O₂. Fluorescence intensity was quantified in 12 random cells per field using FIJI v2.1.0.
Intracellular succinate concentration was measured in cells under normoxic conditions and after exposure to 1- and 6-h hypoxia using a commercial assay (Sigma catalog # MAK184, St. Louis, MO, USA) according to manufacturer instructions. Absorbance at 450 nm was measured using a SpectraMax M3 Microplate Reader (Molecular Devices, San Jose, CA, USA). Succinate concentrations per well were normalized to protein content using a Pierce Rapid Gold Protein Assay (Pierce, Rockford, IL, USA).

Allen K.N., Torres-Velarde J.M., Vazquez J.M., Moreno-Santillán D.D., Sudmant P.H, & Vázquez-Medina J.P. (2024). Hypoxia exposure blunts angiogenic signaling and upregulates the antioxidant system in endothelial cells derived from elephant seals. BMC Biology, 22, 91.

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Antimicrobial Susceptibility Assays

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Susceptibility assays for clinical isolates were performed in complete medium, including 2% (v/v) human AB serum, 5 g/L Albumax II, in 96-well microplates in a final volume of 200 μL, as described previously [13 (link)]. The plates were incubated at 37 °C in an incubation chamber (Billups-Rothenburg Inc) under culture gas for 72 hours, and stored at −20 °C overnight. Microplates were thawed, samples resuspended and, in a duplicate plate, 100 µL from each well was added to 100 μL of SYBR Green lysis buffer (1:5000 SYBR Green I [Thermo Fisher Scientific, S7563], diluted in 20 mM Tris, 5 mM ethylenediaminetetraacetic acid, 0.008% [w/v] saponin, 0.08% [v/v] Triton X-100, pH 7.5). Fluorescence was read in a Spectramax M3 microplate reader (Molecular Devices) at 490 nm excitation and 520 nm emission and dose-response data evaluated as described [13 (link)].

van Schalkwyk D.A., Pratt S., Nolder D., Stewart L.B., Liddy H., Muwanguzi-Karugaba J., Beshir K.B., Britten D., Victory E., Rogers C., Millard J., Brown M., Nabarro L.E., Taylor A., Young B.C., Chiodini P.L, & Sutherland C.J. (2023). Treatment Failure in a UK Malaria Patient Harboring Genetically Variant Plasmodium falciparum From Uganda With Reduced In Vitro Susceptibility to Artemisinin and Lumefantrine. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America, 78(2), 445-452.

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Assessing Glioma Cell Viability

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The viability of the glioma cells transfected with specific oligonucleotides was determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Amresco) uptake method. Briefly, after the cells were incubated for 24 h, they were transfected with specific oligonucleotides, and seeded in a 96-well plate (2 × 10³/well). The cells were incubated for another 48 h in medium containing 10% FBS. MTT solution (10 μl; 5 mg/ml) was added to each well and the plate was incubated for 4 h at 37°C in an incubator. After the medium was removed, the purple formazan crystals were dissolved in 150 μl/well dimethyl sulfoxide (DMSO; Amresco) for 30 min. The absorbance of the formazan was measured at 490 nm on a SpectraMax M3 microplate reader (Molecular Devices, USA).

Han L., Liu D., Li Z., Tian N., Han Z., Wang G., Fu Y., Guo Z., Zhu Z., Du C, & Tian Y. (2015). HOXB1 Is a Tumor Suppressor Gene Regulated by miR-3175 in Glioma. PLoS ONE, 10(11), e0142387.

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Quantifying Inflammatory and Antioxidant Factors

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The COX-2 level was estimated in the cytoplasmic extract, whereas NF-κB p65, NRF-2 and HO-1 levels were estimated in nuclear extract. The nuclear extraction kit was used to separate the cytoplasm from the nucleus following the manufacturer’s instructions. The cell pellet was incubated in 100 µL of pre-extraction buffer on ice for 10 min, vortexed, and centrifuged. The supernatant cytoplasmic extract was collected. The nuclear pellet was incubated in an extraction buffer containing Dithiothreitol (DTT) and Protease Inhibitor Cocktail (PIC) on ice for 15 min. The suspension was centrifuged to obtain the nuclear extract. The protein concentration in the extracts was determined using Bradford’s reagent. The respective ELISA kits were used to measure the levels of COX-2, NF-κB p65, NRF-2 and HO-1. The levels were measured in triplicate. The absorbance was measured using a Spectramax M3 microplate reader (Molecular Devices, LLC., San Jose, CA 95134, USA). The effect of the test compounds on the expression was represented as fold change with reference to the negative control, using the following formula, and the results are presented as mean ± SD (n = 3).

Fold change = \frac{Protein level in LPS E c or compound treated cells}{Protein level in untreated cells}

Mak K.K., Shiming Z., Low J.S., Balijepalli M.K., Sakirolla R., Dinkova-Kostova A.T., Epemolu O., Mohd Z, & Pichika M.R. (2022). Anti-Inflammatory Effects of Auranamide and Patriscabratine—Mechanisms and In Silico Studies. Molecules, 27(15), 4992.

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Quantifying Cellular ATP and Albumin

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Cellular ATP was measured in n=8 individual spheroids per time point from
N=6 preparations (3 per sex) using a CellTiter-Glo Luminescent Cell Viability
Assay (Promega, Madison, WI, USA) according to manufacturer’s
instructions. Media (75 μL out of 100 μL per well) was collected
from same wells used for cellular ATP measurements and stored at −80
°C for albumin measurements. Albumin content in the media was quantified
using a mouse albumin ELISA quantification set (Bethyl Laboratories, Montgomery,
TX, USA) per manufacturer’s instructions as previously described (Norona et al., 2016 (link)). Raw data was
collected using a SpectraMax M3 microplate reader (Molecular Devices, San Jose,
CA, USA). A standard curve was used to calculate ATP (nM) and albumin (ng/mL)
for all samples.

Nautiyal M., Qasem R.J., Fallon J.K., Wolf K.K., Liu J., Dixon D., Smith P.C, & Mosedale M. (2020). Characterization of Primary Mouse Hepatocyte Spheroids as a Model System to Support Investigations of Drug-Induced Liver Injury. Toxicology in vitro : an international journal published in association with BIBRA, 70, 105010.

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Anti-influenza Virus Compound Isolation

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Column chromatography was performed on 200–300 mesh silica gel (Qingdao Marine Chemical Co. Ltd., Qingdao, China). All purifications were monitored by TLC using commercially available glass plates pre-coated with silica gel 60 G (E. Merck Co., Billerica, Germany) and LC-6AD pHPLC (Shimadzu, Kyoto, Japan) with a UV SPD-20A detector using a reversed-phase C18 column (5 μm, 21.2 × 150 mm; Phenomenex Inc., Torrance, CA, USA). All the silica gel and GF₂₅₄ plates were treated with citrate buffer (pH 2.0). The analytical HPLC was performed using an Agilent LC1100 (Agilent Technologies, Santa Clara, CA, USA) equipped with a MWD detector using a reversed-phase C18 column (5 μm, 4.6 × 250 mm; Phenomenex Inc., Torrance, CA, USA). ¹H- and ¹³C-NMR data were all obtained on an Advance DRX-500 spectrometer (Bruker, Rheinstetten, Germany) in CDCl₃ with TMS as an internal standard. HRESIMS was performed on a QTOF-MS (AB SCIEX, Framingham, MA, USA). Anti-influenza virus H5N1 activity was assayed with a SpectraMax M3 microplate reader (Molecular Devices, Sunnyvale, CAUSA).

Wang J., Yan Y.T., Fu S.Z., Peng B., Bao L.L., Zhang Y.L., Hu J.H., Zeng Z.P., Geng D.H, & Gao Z.P. (2017). Anti-Influenza Virus (H5N1) Activity Screening on the Phloroglucinols from Rhizomes of Dryopteris crassirhizoma. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(3), 431.

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Evaluating Cisplatin Liposome Efficacy

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Cells were seeded onto a 96-well plate at a density of 5.0 × 10³ cells per well in 100 μL of medium and incubated overnight (37 °C, 5% CO₂, and 80% humidity). An additional 100 μL of new medium (RPMI medium 1640/10% FBS/1% penicillin-streptomycin) containing either cisplatin or cisplatin liposomes with concentrations ranging from 50 nM to 500 μM Pt equivalents was added to the cells. The tests were conducted in replicates of three for each concentration. After incubation for 72 h, 40 μL of media containing thiazolyl blue tetrazolium bromide solution (2.5 mg/mL) was added. The cells were incubated for 2 h, after which time the medium was carefully removed. To the resulting purple crystals was added 200 μL of DMSO, followed by 25 μL of pH 10.5 glycine buffer (0.1 M glycine/0.1 M NaCl). The optical densities at 570 nm and 690 nm (background) were measured on a SpectraMAX M3 microplate reader (Molecular Devices, Sunnyvale, CA). Wells containing cells that received neither liposome nor drug were considered to represent 100% viability. IC₅₀ values were obtained from sigmoidal fits of semilogarithmic plots of the percentage of viability versus platinum concentration by using Origin 7 SR4 8.0552 software (OriginLab, Northhampton, MA). And had standard deviations between ±10% of the stated IC50 value.

Kieler-Ferguson H.M., Chan D., Sockolosky J., Finney L., Maxey E., Vogt S, & Szoka FC J.r. (2017). Encapsulation, controlled release, and antitumor efficacy of cisplatin delivered in liposomes composed of sterol-modified phospholipids. European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 103, 85-93.

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Serum LPS Concentration Measurement

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Serum LPS concentrations were measured using PYROGENT-5000 kit as per manufacturer’s instructions (Lonza, Mississauga, ON, Canada). The absorbance was measured at 340 nm per minute for 1 h at 37 °C using a SpectraMax^® M3 Microplate Reader (Molecular Devices, LLC. Sunnyvale, CA, USA). Reaction time was defined as the time required for the absorbance to increase 0.03 absorbance units.

Singh V.P., Fontaine M.A., Mangat R., Fouhse J.M., Diane A., Willing B.P, & Proctor S.D. (2021). High Vaccenic Acid Content in Beef Fat Attenuates High Fat and High Carbohydrate Western Diet Induced Changes in Lipid Metabolism and Gut Microbiota in Pigs. Microorganisms, 9(12), 2517.

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MTT Assay for Glioma Cell Viability

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Cell viability was evaluated by using MTT assay. Glioma cells were seeded into 96-well plates at the concentration of 2 × 10³ cells/well. Cells were treated with different concentrations of TMZ (MedChem Express, Monmouth Junction, NJ, USA) for 48 h. Then, 10 µl MTT (5 mg/ml) was added to each well and incubated in the dark at 37℃ for another 4 h. Absorbance was determined at a wavelength of 570 nm using a SpectraMax M3 microplatereader (Molecular Devices, Sunnyvale, CA, USA).

Shangguan W., Lv X, & Tian N. (2019). FoxD2-AS1 is a prognostic factor in glioma and promotes temozolomide resistance in a O6-methylguanine-DNA methyltransferase-dependent manner. The Korean Journal of Physiology & Pharmacology : Official Journal of the Korean Physiological Society and the Korean Society of Pharmacology, 23(6), 475-482.

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Measuring H2O2 Release in Myotubes

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H₂O₂ release from cells was monitored by measuring the conversion of Amplex Red to the highly fluorescent resorufin in the presence of added horseradish peroxidase. A total of 10⁴ cells were incubated in 96-well plates, and after differentiation, the myotubes were treated with 1, 10 and 50 µM pravastatin for 12 h. Cells were incubated in a mixture containing 50 μM Amplex Red reagent (Invitrogen) and 0.1 U/mL horseradish peroxidase in Krebs–Ringer phosphate buffer (in mM: 145 NaCl, 5.7 sodium phosphate, 4.86 KCl, 0.54 CaCl₂, 1.22 MgSO₄, and 22 glucose, pH 7.35). This assay was conducted in the presence and absence of catalase (500 U/mL) for 1 h. Fluorescence was monitored over time in a temperature-controlled (37 °C) SpectraMax M3 Microplate Reader (Molecular Devices) using the excitation and emission wavelengths of 560 and 590 nm, respectively.

Lorza-Gil E., García-Arevalo M., Favero B.C., Gomes-Marcondes M.C, & Oliveira H.C. (2019). Diabetogenic effect of pravastatin is associated with insulin resistance and myotoxicity in hypercholesterolemic mice. Journal of Translational Medicine, 17, 285.

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