Spectramax i3x multi mode microplate reader

Manufactured by Molecular Devices

Sourced in United States, Japan

The SpectraMax i3x Multi-Mode Microplate Reader is a versatile laboratory instrument that can perform various microplate-based assays. It is capable of measuring absorbance, fluorescence, and luminescence within microplates.

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Lab products found in correlation

Celltiter blue cell viability assay, by Promega (4 mentions) 1 step ultra tmb elisa, by Thermo Fisher Scientific (4 mentions) Streptavidin donor bead, by PerkinElmer (4 mentions) Optiplate 384 assay plate, by PerkinElmer (4 mentions) Trizol reagent, by Thermo Fisher Scientific (4 mentions) Alamar blue, by Thermo Fisher Scientific (3 mentions) Cell counting kit 8 (cck8), by Beyotime (3 mentions) Cyquant cell proliferation assay, by Thermo Fisher Scientific (3 mentions) Propidium iodide, by Thermo Fisher Scientific (3 mentions) Vcx130, by Sonics (2 mentions) Passive lysis buffer (plb), by Promega (2 mentions) Dual luciferase reporter assay system, by Promega (2 mentions) Renilla luciferase assay system, by Promega (2 mentions) Immunolon 4 hbx microtiter 96 well strips, by Avantor (2 mentions) Cell counting kit 8 cck 8, by Dojindo Laboratories (2 mentions) Nitrate nitrite colorimetric assay kit, by Cayman Chemical (2 mentions) Caspase glo assay kit, by Promega (2 mentions) Pierce tmb substrate kit, by Thermo Fisher Scientific (2 mentions) Ripa cell lysis buffer, by Thermo Fisher Scientific (2 mentions) Cell counting kit 8 (cck8), by Dojindo Laboratories (2 mentions)

Close spelling variants of this product

Microplate reader (2507 citations) SpectraMax i3x (800 citations) SpectraMax microplate reader (255 citations) SpectraMax i3x microplate reader (128 citations) Multi-mode microplate reader (40 citations) SpectraMax i3x reader (18 citations) I3X microplate reader (4 citations) SpectraMax i3x multi-microplate reader (3 citations) SpectraMax i3x Multi-Mode Microplate (2 citations) SpectraMax i3x Multi-Mode Microplate Reader i3 (2 citations)

182 protocols using spectramax i3x multi mode microplate reader

Cytotoxicity and ATP Evaluation of Formate in HIEC-6 Cells

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The percentage of surviving cells was calculated as follow. HIEC-6 cells (1 × 10⁵/well- 24 well plate) were seeded and maintained as above. After 70% confluence, the cells were treated with Formate (0, 17, 50, 150 mM). After 24, 48, and 72 h of incubation, 50 μl of the supernatant was collected in triplicate to a 96-well plate and stored in −20°C refrigerator for LDH assay. 50 μl of 2% NP40 were added to each well of the 96-well plate, mixed with the supernatant and incubated at room temperature with shaking for 15 min. One hundred microliter of the Cytotoxicity Detection Kit LDH solution (Takara, Japan) was added to each well. After 15 min of incubation at room temperature in the dark, the OD value was measured at 492 nm in a SpectraMax® i3x multi-mode microplate reader (Molecular Devices, LCC. CA, USA) (620 nm as reference).
The CellTiter-Glo® 2.0 reagent was used for determination of ATP content according to the manufacturer's instructions (for G9242 from Promega Biotechnology Company, Madison, WI). Briefly, 100 μl of CellTiter-Glo® 2.0 Reagent were added to each well. The contents were mixed for 2 min on an orbital shaker to induce cell lysis, followed by incubation at room temperature for 10 min to stabilize the luminescent signal. The plates were read using the SpectraMax® i3x multi-mode microplate reader (Molecular Devices, LCC. CA, USA).

Casaburi G., Wei J., Kazi S., Liu J., Wang K., Tao G.Z., Lin P.Y., Dunn J.C., Henrick B.M., Frese S.A, & Sylvester K.G. (2022). Metabolic model of necrotizing enterocolitis in the premature newborn gut resulting from enteric dysbiosis. Frontiers in Pediatrics, 10, 893059.

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Growth-inhibitory effects of combined compounds on neuroblastoma cells

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Growth-inhibitory effects of varying concentrations of 6-thio-dG in combination with etoposide (VP-16, Selleckchem, USA), doxorubicin (Merck, Germany) or ceritinib (LDK378, Selleckchem, USA) on neuroblastoma cell lines with TERT rearrangements (CLB-GA, GI-ME-N), high TERT expression (SH-SY5Y) or MYCN amplification (BE(2)-C, IMR-32, Kelly, LS, TR-14) were determined after 96 h of substance incubation using CellTiter-Glo®. A semi-automated substance screening protocol was established on a Biomek 4000 Automated Workstation (Beckman Coulter, Brea, CA, USA). Single or combined compounds in nine different concentrations and DMSO as control were added in triplicates 24 h after the cells were seeded in a 96-well plate at a density of 3000 cells/well. Cell viability was measured after 96 h of substance incubation using a CellTiter-Glo® (CTG) Luminescent Cell Viability Assay (Promega, Madison, WI, USA). In brief, 100 µl CTG-reagent was added to the wells and incubated for 30 min at room temperature. Signals were measured using a SpectraMax® i3x Multi-Mode microplate reader (Molecular Devices, San Jose, CA, USA). Drug synergy was calculated according to the Chou-Talalay combination index method [17 (link), 18 (link)].

Fischer-Mertens J., Otte F., Roderwieser A., Rosswog C., Kahlert Y., Werr L., Hellmann A.M., Berding M., Chiu B., Bartenhagen C, & Fischer M. (2022). Telomerase-targeting compounds Imetelstat and 6-thio-dG act synergistically with chemotherapy in high-risk neuroblastoma models. Cellular Oncology (Dordrecht), 45(5), 991-1003.

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Protein Detection and Luminescence Assays

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Western blotting was performed following standard procedure. The primary antibody for V5-tag (Abcam, Shanghai, China) and peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, USA) were used for targets detection. The immunoreactive proteins were detected by SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific).
Luciferase assay was performed in the white, opaque 96-well plates using 20 μL of culture supernatant for each well, and 50 μL/well Gluc buffer (100 mM Tris-HCl at pH 8.0, 10 μM coelenterazine, 0.3% Triton X-100, and 70 mM NaI) or Cypridina Glow Assay Buffer containing 1 × Vargulin (Thermo Fisher Scientific) was added for Gluc or hCluc assay. Total chemiluminescence was acquired using the SpectraMax i3x multi-mode microplate reader (Molecular Devices). Fluorescence intensities in cells were measured using the same microplate reader.

Bei Z.C., Yu H., Wang H., Li Q., Wang B., Zhang D., Xu L., Zhao L., Dong S, & Song Y. (2023). Orthogonal dual reporter-based gain-of-signal assay for probing SARS-CoV-2 3CL protease activity in living cells: inhibitor identification and mutation investigation. Emerging Microbes & Infections, 12(1), 2211688.

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Ultrasonication-Assisted Synthesis of Trehalose-Conjugated Nanogels

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Ultrasonication with Sonics VCX 130 (diameter of probe: 3 mm, Sonics & Materials, Inc., USA) was carried out during preparation of a miniemulsion (before photo-polymerization) and redispersion of nanogel powder at amplitudes of 60% (5 min) and 40% (30 s), respectively. Each purification process was followed by dialysis and lyophilization in a freeze dryer (ALPHA 1–2 LDplus, CHRIST). NMR spectra were recorded in deuterated solvents (Deutero GmbH) with internal standards using an NMR spectrometer operating at 600 MHz (Varian). Fluorescence study was conducted in SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices, USA). Both the amount of conjugated trehalose and trehalose release profile were determined enzymatically using a Trehalose Assay Kit (Megazyme International, Ireland). Phosphate buffered saline (PBS, pH 7.0 and 7.4), potassium chloride (KCl), and normal saline (NS) solutions. Deionized water (DI water) was produced using a reverse osmosis system (conductivity < 2 µS/cm).

Zhong Y., Maruf A., Qu K., Milewska M., Wandzik I., Mou N., Cao Y, & Wu W. (2023). Nanogels with covalently bound and releasable trehalose for autophagy stimulation in atherosclerosis. Journal of Nanobiotechnology, 21, 472.

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Evaluating mHippoE-18 Cell Proliferation with CH/NGF-μS/PCL Conduit

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To determine the influence of CH/NGF-µS/PCL conduit on mHippoE-18 cell proliferation, we performed an assay where 0.2 mL of hippocampal cell suspension (5 × 10⁴ cells/mL) was seeded in a 96-well tissue culture plate (Corning, Wilmington, NC, USA); then, after 24 h of incubation, the conduit fragments constituting 1/10 of the well surface were placed carefully on the cell monolayer. After another 24 h of incubation, DNA content was measured using a CyQUANT^® Cell Proliferation Assay Kit (Thermo Fisher Scientific, Waltham, Massachusetts). The wells with samples were washed with PBS and then frozen at −80 °C. Next the plate was thawed at room temperature, and samples were lysed in buffer containing the CyQuant-GR dye, which bound to cellular nucleic acids. Fluorescence was measured using SpectraMax^® i3x Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA) at E_m = 520 nm and E_x = 480 nm. The commercially available biomaterial Safety-Lok™ blood collection (BCS) served as a negative control.

Nawrotek K., Kubicka M., Gatkowska J., Wieczorek M., Michlewska S., Bekier A., Wach R, & Rudnicka K. (2022). Controlling the Spatiotemporal Release of Nerve Growth Factor by Chitosan/Polycaprolactone Conduits for Use in Peripheral Nerve Regeneration. International Journal of Molecular Sciences, 23(5), 2852.

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Mycobacterium tuberculosis Coenzyme A Biosynthesis

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M. tuberculosis H37RvMA and its coaBC Tet-OFF conditional knockdown derivative^{12 (link)} were grown in Difco Middlebrook 7H9 broth (BD) supplemented with Middlebrook albumin-dextrose-catalase (ADC) enrichment (BD), 0.2% glycerol (Sigma-Aldrich) and 0.05% Tween-80, unless otherwise indicated. Hygromycin and kanamycin were used at final concentrations of 50 μg/mL and 25 μg/mL, respectively, and pantethine (Sigma-Aldrich) supplementation was included at 2.5 mg/mL where required. The anhydrotetracycline (ATc) inducer was used at concentrations up to 200 ng/mL in order to transcriptionally silence coaBC in the Tet-OFF hypomorph.
The minimum inhibitory concentrations (MIC₉₉) of the compounds were determined by measuring fluorescence output using Alamar Blue, as previously described (Singh et al.^{56 (link)}). Briefly, 2-fold serial dilutions of compound were inoculated with a suspension of M. tuberculosis at a cell density of ~10⁵ CFU/mL in a 96-well microtiter plate and incubated at 37 °C for 10 days, following which 10 μL Alamar Blue solution was added and the plates were incubated for a further 24 h. Fluorescence as an indication of growth was measured using a SpectraMax i3x Multi-Mode Microplate Reader (Molecular Devices) in bottom-reading mode with excitation at 544 nm and emission at 590 nm.

Mendes V., Green S.R., Evans J.C., Hess J., Blaszczyk M., Spry C., Bryant O., Cory-Wright J., Chan D.S., Torres P.H., Wang Z., Nahiyaan N., O’Neill S., Damerow S., Post J., Bayliss T., Lynch S.L., Coyne A.G., Ray P.C., Abell C., Rhee K.Y., Boshoff H.I., Barry CE I.I.I., Mizrahi V., Wyatt P.G, & Blundell T.L. (2021). Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site. Nature Communications, 12, 143.

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Neurotoxicity Assays in Astrocyte-LUHMES Coculture

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Coculture of Mn-treated EV-/REST-transfected astrocytes and differentiated LUHMES cells were treated with toxic concentrations of glutamate (250 μM, 12 h). Endpoint product fluorescence was measured in each assay using the Spectramax i3x Multi-Mode microplate reader from Molecular Devices. To measure cell viability and apoptosis, 10 μl of resazurin (5 mg/ml in PBS) or annexin V was added, and cells were incubated for 15 min at 37 °C, followed by measurement of fluorescence intensity at excitation/emission wavelength of 530/590 nm for resazurin and 494/518 for annexin V–FITC. Ca²⁺ influx as an indicator of neuronal excitation and activation was measured using Life Technologies Fluo-4AM. Mitochondrial Δψm as an indicator of mitochondrial health and functional status was measured using the Life Technologies tetramethylrhodamine ethyl ester. Generation of ROS as an indicator of oxidative stress was measured using the Life Technologies ROS probe chloromethyl derivative of 2′,7′-dichlorodihydrofluorescein diacetate. Endpoint fluorescence was determined at an excitation/emission wavelength of 494/506 nm (for Ca²⁺ influx), 549/575 nm (for Δψm), and 485/527 nm (for ROS).

Pajarillo E., Digman A., Nyarko-Danquah I., Son D.S., Soliman K.F., Aschner M, & Lee E. (2021). Astrocytic transcription factor REST upregulates glutamate transporter EAAT2, protecting dopaminergic neurons from manganese-induced excitotoxicity. The Journal of Biological Chemistry, 297(6), 101372.

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Measuring Intracellular ROS Levels

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Levels of intracellular ROS were determined using the cell-permeant fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probes^®, ThermoFisher Scientific, Waltham, MA, USA). Upon cleavage of the acetate groups by intracellular esterases and oxidation, the nonfluorescent H2DCFDA is converted to the highly fluorescent 2′,7′-dichlorofluorescein (DCF). Elicited murine peritoneal macrophages were plated in a 96-well plate (2 × 10⁴ cells/well) and exposed to various concentrations of sterile UICC crocidolite (SPI Supplies) asbestos fibers (0, 1, 5, 10, and 20 µg/cm²) and LGM2605 (0, 10, 25, 50, and 100 µM). The fluorescence intensity was then measured on a SpectraMax i3x Multi-Mode microplate reader (Molecular Devices, Sunnyvale, CA, USA) using an excitation wavelength in the range of 492–495 nm and a fluorescence emission detection at 517–527 nm.

Pietrofesa R.A., Velalopoulou A., Albelda S.M, & Christofidou-Solomidou M. (2016). Asbestos Induces Oxidative Stress and Activation of Nrf2 Signaling in Murine Macrophages: Chemopreventive Role of the Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605). International Journal of Molecular Sciences, 17(3), 322.

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Cell Viability Assay Using CellTiter-Blue

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The CellTiter-Blue® Cell Viability assay was acquired from Promega Corporation (Madison, WI, USA). The cell viability assay was performed as follows: the cells (10⁴/well) were incubated with a concentration gradient of the test compounds ranging from 0.01 to 100 μM, in 96-well plates for 48 h at 37°C. Subsequently, 20 µL of the CellTiter-Blue® Cell Viability reagent was added to each well and incubated for an additional 2 h for the development of fluorescence. The fluorescence emission was measured at 590 nm using the SpectraMax® i3x Multi-Mode Microplate Reader (Molecular Devices, LLC; San Jose, CA, USA.) and plotted against the drug concentrations to determine the IC₅₀ of the test compounds.

Khayat M.T., Omar A.M., Ahmed F., Khan M.I., Ibrahim S.M., Muhammad Y.A., Malebari A.M., Neamatallah T, & El-Araby M.E. (2022). Insights on Cancer Cell Inhibition, Subcellular Activities, and Kinase Profile of Phenylacetamides Pending 1H-Imidazol-5-One Variants. Frontiers in Pharmacology, 12, 794325.

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Investigating miR-106-5p and KLF15 Interaction

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To determine the interaction between miR-106-5p and its potential target gene (KLF15), 293 T cells and DF1 cells were seeded in 24-well plates and co-transfected in triplicate with 80 nmol/L miR-106-5p agomir or agomir NC and 500 ng of the aforementioned wild-type or mutant plasmids per well. At 48 h post-transfection, the cells were washed thrice with PBS (Gibco) and lysed with 1× passive lysis buffer (Promega, Madison, WI, USA) for 15 min. Firefly luciferase and Renilla luciferase signals were measured using the Dual-Luciferase® Reporter Assay System (Promega, Madison, WI, USA) on a SpectraMax® i3x Multi-Mode Microplate Reader (Molecular Devices Corporation, Sunnyvale, CA). The Renilla luciferase signal was normalized to the firefly luciferase signal. All reactions were performed in triplicate.

Tian W., Hao X., Nie R., Ling Y., Zhang B., Zhang H, & Wu C. (2022). Integrative analysis of miRNA and mRNA profiles reveals that gga-miR-106-5p inhibits adipogenesis by targeting the KLF15 gene in chickens. Journal of Animal Science and Biotechnology, 13, 81.

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