Spectramax gemini em

Manufactured by Molecular Devices

Sourced in United States, United Kingdom

The SpectraMax Gemini EM is a multimode microplate reader capable of absorbance, fluorescence, and luminescence detection. It features a xenon flash lamp, dual-monochromator optics, and a photomultiplier tube detector. The instrument is designed to enable a wide range of applications in life science research, including cell-based assays, enzyme kinetics, and nucleic acid quantification.

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Close spelling variants of this product

Fluorimeter SpectraMax Gemini EM SpectraMax Gemini EM Fluorescence Microplate Reader SpectraMax Gemini EM plate reader SpectraMax GEMINI EM microplate spectrofluorometer SpectraMax Gemini EM Microplate Reader SpectraMax Gemini XPS/EM Plate Reader SpectraMax Gemini XPS/EM Spectramax II Gemini EM plate reader SpectraMax Gemini EM Fluorescence Reader Spectramax Gemini EM spectrofluorometer

246 protocols using spectramax gemini em

Oxidative Stress-Induced GPx and TBARS

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To investigate the effects of oxidative stress induced by H 2 O 2 , we measured glutathione peroxidase (GPx) activity and thiobarbituric acid reactive substances (TBARS) in IEC-18 cells. Both were normalized to protein, measured by BCA protein assay kit (Thermo Scientific, USA). GPX activity in cell lysate was measured using a commercial test kit (Cayman Chemical Company, Michigan) according to the manufacturer's instructions. Briefly, GPx catalyzed the oxidation of glutathione (GSH) by hydroperoxide. In the presence of glutathione reductase and NAPDH, the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NAPDH to NAPD+. The decreased in absorbance at 340nm was measured by a micro-plate reader (Molecular Devices SpectraMax Gemini EM). The specific activity for GPx was expressed in units per milligram protein (U/mg protein).
TBARS were measured in the cell lysate (Cayman Chemical Company, Michigan).
Briefly, tricholoroacetic in sodium sulfate was added into cell lysates. After precipitation, the samples were washed by sulfuric acid and then incubated in a boiling water bath for one hour. After cooling, the samples were extracted with nbutanol and centrifuged for 10 minutes at 1600 x g at 4°C. Absorbance was measured using a micro-plate reader (Molecular Devices SpectraMax Gemini EM) at a wavelength of 540 nm.

Li B., Zani A., Martin Z., Lee C., Zani-Ruttenstock E., Eaton S, & Pierro A. (2016). Intestinal epithelial cell injury is rescued by hydrogen sulfide. Journal of pediatric surgery, 51(5).

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Mitochondrial Dysfunction Evaluation

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Harvested SKNMC cells the day before the experiment and seeded onto 6-well plate with 2 × 10⁵ cells per well in culture media and allowed to attach overnight. The cells were pretreated with the doses of PPEES at 50, 100 and 200 µg/mL at 37 °C for 6 h and washed with PBS. Then, the cells were treated with Mn (500 µM) for an additional 24 h. Finally, after washing, the cells were seeded on a 6-well plate with PBS once and incubated with JC-1 (10 mM final concentration) for 30 min at 37 °C in the dark. The JC-1 green fluorescence intensity was measured in the microplate reader (Spectra MAX, Gemini EM, Molecular Device) at an excitation wave length of 488 nm and an emission wave length of 530 nm after the cells were washed two times with PBS to remove the extracellular JC-1. Monomeric JC-1 green fluorescence emission and aggregate were measured at excitation wavelength 488 nm, emission wavelength 530 nm on a microplate reader (Spectra MAX, Gemini EM, Molecular Device, Sunnyvale, CA, USA).

Bahar E., Lee G.H., Bhattarai K.R., Lee H.Y., Choi M.K., Rashid H.O., Kim J.Y., Chae H.J, & Yoon H. (2017). Polyphenolic Extract of Euphorbia supina Attenuates Manganese-Induced Neurotoxicity by Enhancing Antioxidant Activity through Regulation of ER Stress and ER Stress-Mediated Apoptosis. International Journal of Molecular Sciences, 18(2), 300.

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Caspase-3 Activity and Cell Viability Assays

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Caspase-3 activity in HeLa cells was determined by assaying DEVDase activity in cell lysates using the ApoTarget caspase 3 / CPP32 fluorimetric protease assay kit (Biosource) according to the manufacturer’s instructions. Fluorescence was measured using a SpectraMax Gemini EM plate reader (Molecular Devices). NPC fibroblasts were stained with Hoechst (Immunocytochemistry Technologies). Cells were counted in five randomly selected fields per transfection at 200x magnification and scored for chromatin condensation. The viability of primary mouse cortical neurons and HeLa cells was determined by XTT assay (Cell Proliferation Kit II, Roche). XTT reagent and activation reagent were mixed at a ratio of 50:1 and added to cultures. After incubating for 4 hrs at 37°C, absorbance at 490 nm and 650 nm was measured using a SpectraMax Gemini EM plate reader (Molecular Devices).

Chung C., Elrick M.J., Dell’Orco J.M., Qin Z.S., Kalyana-Sundaram S., Chinnaiyan A.M., Shakkottai V.G, & Lieberman A.P. (2016). Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease. PLoS Genetics, 12(5), e1006042.

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Evaluating Biocompatibility with Resazurin Assay

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A resazurin assay was used in order to evaluate the systems’ biocompatibility. Human fibroblasts and Raw 264.7 cells were seeded in 96-well plates at densities of 1 × 10⁴ cells/well and 0.8 × 10⁴ cells/well, respectively. For human fibroblasts, 0.3 µg of encapsulated mcDNA from each system was added per well. Raw 264.7 cells were transfected by adding 0.24 µg of encapsulated mcDNA from each system per well. After 24 and 48 h of transfection, the culture medium was discarded and 100 µL of fresh complete medium and 20 µL of resazurin 0.1% (w/v) were added to each well and incubated over four hours in the dark at 37 °C in a humidified atmosphere of 5% CO₂. After incubation, the fluorescence was measured in a spectrofluorometer (SpectraMAX^® GeminiTM EM, Molecular Devices, San Jose, CA, USA) at an excitation wavelength of 544 nm and emission wavelength of 590 nm to analyze the resorufin fluorescence produced.

Serra A.S., Eusébio D., Neves A.R., Albuquerque T., Bhatt H., Biswas S., Costa D, & Sousa Â. (2021). Synthesis and Characterization of Mannosylated Formulations to Deliver a Minicircle DNA Vaccine. Pharmaceutics, 13(5), 673.

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Nanoparticle Biocompatibility Assessment

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To evaluate the nanoparticle’s biocompatibility, a resazurin assay was used. This assay was applied in two different cell lines, hFibro and JAWSII cells. Cells were seeded in 96-well plates with a density of 1 × 10⁴ cells/well. Transfections with different chitosan systems and naked pDNA-E7mut were performed for 48 and 72 h. After, the culture medium was discarded and 100 µL of fresh complete medium and 20 µL of resazurin 0.1% (w/w) were added to each well. Cells were incubated in the dark over 4 h at 37 °C in a humidified atmosphere containing 5% CO₂. Finally, the resorufin fluorescence was measured in a spectrofluorometer (SpectraMAX^® GeminiTM EM, Molecular Devices, San Jose, CA, USA) by defining an excitation wavelength of 544 nm and an emission wavelength of 590 nm. Non-transfected cells were used as negative control and cells treated with 70% ethanol were used as the positive control. The statistical analysis was performed with two-way ANOVA followed by Tukey test (GraphPad Prism 9 software, San Diego, CA, USA).

Rodolfo C., Eusébio D., Ventura C., Nunes R., Florindo H.F., Costa D, & Sousa Â. (2021). Design of Experiments to Achieve an Efficient Chitosan-Based DNA Vaccine Delivery System. Pharmaceutics, 13(9), 1369.

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Quantification of Neutrophil Extracellular DNA

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Neutrophil supernatant was collected and measured using Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) according to the manufacturer's protocol. Briefly, isolated neutrophils (1 × 10 6 cells/well) were seeded into 96-well plates in 100 μL of RPMI 1640 medium (phenol-red-free) and incubated with 10 mM NAC, 1 μM thapsigargin, or 50 μM 2APB for 5 min, respectively, then stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA) for 3 h. Then the aqueous working solution of the Quant-iT PicoGreen reagent was added to each well. Subsequently, fluorescence intensity was measured via a Fluorescence Spectrophotometer (Spec-traMax Gemini EM, Molecular Devices) at 520 nm after excitation at 485 nm. The level of extracellular DNA was quantified to assess the ability of neutrophils to produce NET.

Zhang B., Ma X., Loor J.J., Jiang Q., Guo H., Zhang W., Li M., Lv X., Yin Y., Wen J., Wang J., Xu C, & Yang W. (2022). Role of ORAI calcium release-activated calcium modulator 1 (ORAI1) on neutrophil extracellular trap formation in dairy cows with subclinical hypocalcemia. Journal of dairy science, 105(4).

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Preparation and Characterization of Advanced Glycation End-products

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The AGEs were prepared as previously described by Xu et al (21 (link)). Briefly, BSA was incubated under sterile conditions with 50 mM D-glucose in 5% CO₂/95% air at 37°C in the dark for 12 weeks. The unincorporated glucose molecules were removed by dialysis overnight against 0.01 M phosphate-buffered saline (PBS). The success of the AGE preparation was determined using a Spectra Max Gemini EM fluorescence reader (Molecular Devices LLC, Sunnyvale, CA, USA). The measurements were performed in triplicate at the excitation wavelength of 370 nm, and the emission peak was observed at 460 nm. The AGEs were stored at −20°C until use. BSA incubated without D-glucose under the same conditions was used as the negative control.

Xie Y., Yu D., Wu J, & Li L. (2017). Protective effects of physiological testosterone on advanced glycation end product-induced injury in human endothelial cells. Molecular Medicine Reports, 15(3), 1165-1171.

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Antiglycation Potential of Millets and Sorghum

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BSA-MGO mediated middle-stage antiglycation activity of the selected millet and sorghum samples was determined according to the methods of Abeysekera et al. and Lunceford and Gugliucci [30 (link), 31 (link)] with some modifications. A reaction volume of 1 ml containing 10 mg/ml BSA, 5 mM MGO, and different concentrations of millet and sorghum samples (50, 100, 200, 400 μg/ml; n = 4 each) in 0.1 M phosphate buffer (pH 7.4) containing 0.02% sodium azide were incubated at 37°C for 6 days. Then, fluorescence intensity was measured at an excitation and emission wave lengths of 370 nm and 440 nm, respectively, using a florescence 96-well microplate reader (SpectraMax, Gemini EM, Molecular Devices, Inc., USA). Rutin (6.25, 12.5, 25, 50, 100, 200 μg/ml) was used as the positive control. Results were expressed as % middle-stage antiglycation activity and IC₅₀ values.

Senevirathne I.G., Abeysekera W.K., Abeysekera W.P., Jayanath N.Y., Galbada Arachchige S.P, & Wijewardana D.C. (2021). Antiamylase, Antiglucosidase, and Antiglycation Properties of Millets and Sorghum from Sri Lanka. Evidence-based Complementary and Alternative Medicine : eCAM, 2021, 5834915.

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Paraquat and Lectin Effects on Alpha-Synuclein Fibrillation

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To examine the effect of paraquat and lectin on the kinetics of fibril formation, an in vitro fibrillation assay was used, as described previously.^{53 (link)} Briefly, solutions containing: (i) purified recombinant α-synuclein alone (35 µM in 50 mM Tris-HCl buffer, pH 7.5); (ii) lectin from Pisum sativum (0.0025%); (iii) paraquat (100 µM); or (iv) a combination of lectin and paraquat, were incubated at 37 °C with constant shaking at 300 rpm for ~40 h. Each sample was plated in triplicate on a 96-well plate, and 20 µM Thioflavine T, a fluorescent dye that binds to fibrillary structures, was added. The fluorescence (excitation at 450 nm and emission at 485 nm) was measured at different time points using a fluorescence plate reader (Spectramax Gemini EM, Molecular Devices, Sunnyvale, CA) interfaced with Softmax® pro 6.3.1 software (Molecular Devices). The relative fluorescence units were averaged and plotted as a function of time; the resulting plot was interpolated, normalized and fitted to a sigmoidal curve using GraphPad Prism® software (GraphPad Software, LaJolla, CA, USA).

Anselmi L., Bove C., Coleman F.H., Le K., Subramanian M.P., Venkiteswaran K., Subramanian T, & Travagli R.A. (2018). Ingestion of subthreshold doses of environmental toxins induces ascending Parkinsonism in the rat. NPJ Parkinson's Disease, 4, 30.

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ROS Quantification in C. elegans Larvae

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The generation of ROS was compared between L1 and L4 larvae stages of NS worms, which were treated with 1, 10, or 100 ng/mL STX for 180 min. More than 100 animals per sample were transferred into the wells of a black 96-well plate containing 100 μL M9 buffer. Dichlorofluorescein diacetate (DCFDA; 50 mM in M9 buffer, 100 mL; Invitrogen) was added to each well. ROS-associated fluorescence levels were measured kinetically with a fluorescence plate reader (Spectra-Max Gemini EM; Molecular Devices, Sunnyvale, CA, USA) at 485 nm excitation and 520 nm emission wavelengths at room temperature, every 2 min for 3 h. Data were normalized to those for bacterial controls composed of 100 mL E. coli HT115 (DE3) expressing dsRNA against prdx-3 or an empty vector containing M9 buffer in separate wells of the same plate. In cells, DCFDA is deacetylated by endogenous esterases to dichlorofluorescein, which reacts with ROS and generates the fluorophore DCF. Although the specific ROS responsible for DCFDA fluorescence is uncertain, DCFDA can be used in whole C. elegans as a marker of general ROS production [79 (link),80 (link)]. Three independent studies were performed.

Wu H., Prithiviraj B, & Tan Z. (2023). Physiological Effects of Oxidative Stress Caused by Saxitoxin in the Nematode Caenorhabditis elegans. Marine Drugs, 21(10), 544.

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