Spectramax5

Manufactured by Molecular Devices

Sourced in United States

The SpectraMax5 is a high-performance multimode microplate reader designed for a wide range of applications in life science research and drug discovery. It offers accurate and reliable absorbance, fluorescence, and luminescence detection across a broad wavelength range. The SpectraMax5 is capable of performing various microplate-based assays, including cell-based, biochemical, and molecular biology experiments.

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Lab products found in correlation

Flow cytometry, by Nikon (1 mentions) Bright glo luciferase assay system, by Promega (1 mentions) Quercetin, by Merck Group (1 mentions) Pierce ldh cytotoxicity assay, by Thermo Fisher Scientific (1 mentions) 1 oleoyl lysophosphatidic acid lpa, by Cayman Chemical (1 mentions) Rhoa g lisa, by Cytoskeleton (1 mentions) Rosetta 2 de3 cells, by Merck Group (1 mentions) Victor x3 multilabel plate reader, by PerkinElmer (1 mentions) Spectra max 5 plate reader, by Molecular Devices (1 mentions) Formamide, by Merck Group (1 mentions) Evans blue, by Merck Group (1 mentions) Sod assay kit, by Nanjing Jiancheng (1 mentions) Mda assay kit, by Beyotime (1 mentions) Mpo assay kit, by Nanjing Jiancheng (1 mentions) Rapid abts method, by Beyotime (1 mentions) Mitosox, by Thermo Fisher Scientific (1 mentions) Atp bioluminescence assay kit, by Beyotime (1 mentions) Gsh gssg glo assay, by Promega (1 mentions) Cell counting kit 8 cck 8, by AbMole (1 mentions) Cellrox green, by Thermo Fisher Scientific (1 mentions)

29 protocols using spectramax5

Cytotoxicity and Proliferation Assays for hCMPCs

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Cytotoxicity was assessed with the Cell Counting Kit-8 (CCK-8, Dojin, Japan). hCMPCs were transfected for 48 h, and 10 µl of the CCK-8 solution was added to each well of the plate. After incubation for 2 h at 37°C, the absorbance was measured at 450 nm using the Spectra Max5 (Molecular Devices, USA).
Cell Proliferation ELISA combined with BrdU (Colorimetric) (11647229001, Roche, Switzerland) were used to evaluate hCMPC proliferation. hCMPCs were separately transfected with miR-10a mimics (50 nM) or inhibitor (50 nM) for 48 h. BrdU was added to the growth medium, and the mixture was incubated overnight. After removing the growth medium, cells were fixed and denatured using FixDenat for 30 min. Then, anti-BrdU-POD that binds the BrdU incorporated in the newly synthesized DNA was added. Finally, substrate was introduced, and the absorbance at 370 nm and 492 nm (reference) was detected with the Spectra Max5 (Molecular Devices, USA). The proliferation rate was further verified using the EdU assay (C10339, Invitrogen, USA). The cell counting experiment was also performed to measure hCMPCs proliferation with crystal violet staining (Biotime, China).

Liang D., Zhen L., Yuan T., Huang J., Deng F., W.u.y.a.h.a.n., Zhang H., Pan L., Liu Y., The E., Yu Z., Zhu W., Zhang Y., Li L., Peng L., Li J, & Chen Y.H. (2014). miR-10a Regulates Proliferation of Human Cardiomyocyte Progenitor Cells by Targeting GATA6. PLoS ONE, 9(7), e103097.

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MTT Assay for Cell Viability

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MTT (3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide) assay is the most commonly used assay to evaluate cell proliferation and viability [21 ]. Proliferating cells express higher metabolic activity, converting MTT into a purple-colored formazan product with an absorbance maximum near 570 nm. For this assay, cells were cultured as described above (section cell culture) for a period of 1, 3, and 7 days (n = 5/time point). Standard MTT cell proliferation assay [22 (link)] was performed for the three Ti substrates. Tissue culture plastic was used as control. The optical density was measured at absorbance = 570 nm using a spectrophotometer (SpectraMax5, Molecular Devices, USA).

Subramani K., Pandruvada S.N., Puleo D.A., Hartsfield JK J.r, & Huja S.S. (2016). In vitro evaluation of osteoblast responses to carbon nanotube-coated titanium surfaces. Progress in Orthodontics, 17, 23.

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NQO1 Enzyme Kinetics Assay

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Initial rates of NQO1 substrate digestion (0.4–25 μM) were monitored using an assay in which the oxidation of NADPH to NADP+ was quantified at 340 nm at 30^oC using Spectramax 5 (Molecular Devices). Reactions of 0.02 μM NQO1, 800 μM NADPH in 50 mM potassium phosphate (pH 7.4), and 5% DMSO with or without 5 mM dicoumarol were initiated by addition of NADPH. Wells were monitored every 3 seconds for 2 minutes to obtain an initial linear signal that was converted to “μM NADPH per minute per μM NQO1” using a standard curve. Michaelis-Menten curves were generated with GraphPad Prism 5. Reactions were performed in triplicate. Similar reactions were carried out with purified NADPH:cytochrome P-450 reductase (POR) (C8113, Sigma), carbonyl reductase 1 (CBR1) (ab85336, Abcam), and thioredoxin (TRX1) (ab51064, Abcam).

Froeling F.E., Swamynathan M.M., Deschênes A., Chio I.I., Brosnan E., Yao M.A., Alagesan P., Lucito M., Li J., Chang A.Y., Trotman L.C., Belleau P., Park Y., Rogoff H.A., Watson J.D, & Tuveson D.A. (2019). Bioactivation of napabucasin triggers reactive oxygen species–mediated cancer cell death. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(23), 7162-7174.

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Pseudovirus Titration Protocols for 293T-ACE2 Cells

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For titration with luciferase assay, 2 × 104 293T-ACE2 or 293T-H-aff-ACE2 cells were seeded in 96-well plates. Serially diluted pseudovirus supernatants were mixed with polybrene in DMEM at a concentration of 10 μg/mL in a total volume of 100 μL. The mixture was then added to each well. After 24, 48, or 72 h incubation at 37°C, the medium was removed. Luciferase activity was assayed by using the Bright-Glo Luciferase Assay System (Promega, Madison, WI, USA) and measured with the Spectramax 5 plate reader (Molecular Devices, San Jose, CA, USA). IU per mL was calculated by this formula: (infected cell readings − non-infected cell readings) × dilution factor.
For titration with GFP count, 1 × 105 293T-ACE2 cells were seeded in 24-well plates. When cells were seeded, serially diluted pseudovirus supernatants were mixed with polybrene in DMEM at a concentration of 10 μg/mL in a total volume of 1 mL. The mixture was then added to each well. After 72 h incubation at 37°C, the cells were imaged under Nikon inverted fluorescent microscopy (Melville, NY, USA), and the number of GFP⁺ cells was then quantified by flow cytometry (BD Biosciences, San Jose, CA, USA).

Fu X., Tao L, & Zhang X. (2020). Comprehensive and systemic optimization for improving the yield of SARS-CoV-2 spike pseudotyped virus. Molecular Therapy. Methods & Clinical Development, 20, 350-356.

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Quantifying Polyphenols in Plant Extracts

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The Folin-Ciocalteu method was used as previously described [24 ], and later modified by Harris et al. (2008). A standard curve was constructed using dilutions of quercetin (Sigma Aldrich Co) at concentrations of 1.0, 0.75, 0.5, 0.25, 0.10, and 0.05 mg/mL. Eight hundred micro liters of Folin reagent was added to 160 μL of sample with 540 μL 7.5% NaHCO₃ and stored in the dark for 2 h. Absorbance was recorded at 725 nm using a spectrophotometer (Molecular Devices SpectraMax 5, Molecular Devices, Sunnyvale, CA, USA). Fifteen milligrams of crude extract was re-solubilized in 80% ethanol at 10 mg/mL inside 1.5 mL centrifuge tubes. The samples were then vortexed until totally dissolved and sonicated for 15 min. These extracts were dyed using the same procedure and the concentration determined using the standard curve.

Downing A.D., Eid H.M., Tang A., Ahmed F., Harris C.S., Haddad P.S., Johns T., Arnason J.T., Bennett S.A, & Cuerrier A. (2019). Growth environment and organ specific variation in in-vitro cytoprotective activities of Picea mariana in PC12 cells exposed to glucose toxicity: a plant used for treatment of diabetes symptoms by the Cree of Eeyou Istchee (Quebec, Canada). BMC Complementary and Alternative Medicine, 19, 137.

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Cytotoxicity Evaluation by LDH Assay

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Toxicity was determined by measuring the cytoplasmic lactate dehydrogenase enzyme (LDH) using Pierce LDH cytotoxicity assay (Life Technologies, Carlsbad, CA) in the media at end point following the instructions of the manufacturer. Absorbance was measured using a microplate reader (SpectraMax5, Molecular Devices, San Jose, CA).

Subramanian P, & Becerra S.P. (2019). Role of the PNPLA2 Gene in the Regulation of Oxidative Stress Damage of RPE. Advances in experimental medicine and biology, 1185, 377-382.

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RhoA Activation Assay in 3D Spheroids

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RhoA G-LISA (Cytoskeleton, Inc) was performed according to the manufacture's specifications. Briefly, monolayer cells were grown to confluence before being stimulated with 10 µM 1-Oleoyl Lysophosphatidic acid LPA (Cayman Chemical) for 5 minutes. Cells were collected using the lysis buffer from the kit. A minimum of 200 spheroids were collected at 48, 72, 96, and 120hr. Spheroids were collected on ice, centrifuged briefly to pellet. Lysis buffer was added and the spheroids were dissociated before starting the assay. Each condition was testing in triplicate and the experiment was performed thrice. Standard deviations (SD) were expressed from at least triplicate wells for a representative experiment. Luminescence was measured on the SpectraMax5 (Molecular Devices).

Pettee K.M., Dvorak K.M., Nestor-Kalinoski A.L, & Eisenmann K.M. (2014). An mDia2/ROCK Signaling Axis Regulates Invasive Egress from Epithelial Ovarian Cancer Spheroids. PLoS ONE, 9(2), e90371.

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Cytotoxicity Assay for Cell Lines

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Cells were seeded in 96-well plates (1.3 × 10⁴ cells/well) and incubated at 37 °C for 50 h to allow them to reach confluence. Culture media were replaced with serum-free media containing NaIO₃ or H₂O₂ at indicated concentrations and incubated at 37 °C for 16 h. Toxicity was determined by measuring the cytoplasmic lactate dehydrogenase enzyme (LDH cytotoxicity assay, Pierce™, Life Technologies, Carlsbad, CA) in conditioned media following the instructions of the manufacturer. The absorbance was measured using a microplate reader (SpectraMax5, Molecular Devices, Downingtown, PA).

Nadal-Nicolas F.M, & Patricia Becerra S. (2018). Pigment Epithelium-derived Factor Protects Retinal Pigment Epithelial Cells Against Cytotoxicity “In Vitro”. Advances in experimental medicine and biology, 1074, 457-464.

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Characterizing Gene Expression in E. coli

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Rosetta 2(DE3) cells (Millipore Corporation) were grown in Lysogeny broth (LB) medium. Cells co-transformed with the reporter and regulator plasmids were grown overnight in LB medium at 37°C, 225 rpm. Next morning, 2 ml of fresh LB medium was inoculated with 50-μl overnight culture in a 24-well deep well plate covered by a gas permeable film and grown at 37°C, 225 rpm for 6 h (Optical density of the culture at 600 nm (OD₆₀₀) was about 0.5). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added into each well to a final concentration of 0.5 mM and the cells were grown for additional 2.5 h. Optical density was read at 600 nm. For monocistronic experiments, GFP fluorescence was measured using VICTOR X3 Multilabel Plate Reader (PerkinElmer) and the excitation and emission wavelengths for GFP were set at 485/528 nm. For bicistronic experiments, GFP and mCherry fluorescence was measured using SpectraMax 5 (Molecular Devices), and the excitation and emission wavelengths for GFP were set at 485/538 nm and the excitation and emission wavelengths for mCherry were set at 584/612 nm. All experiments were performed in triplicates.

Cao J., Arha M., Sudrik C., Mukherjee A., Wu X, & Kane R.S. (2015). A universal strategy for regulating mRNA translation in prokaryotic and eukaryotic cells. Nucleic Acids Research, 43(8), 4353-4362.

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Mycobacterium tuberculosis Growth Assay

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Strain H37Rv was grown to mid log phase in standard 7H9 media. The culture was then diluted to an OD₆₀₀ of 0.01 and aliquoted in a 96-well plate with the final concentrations of m-toluate as indicated in the respective figure. Ethanol carrier concentration was equal in all conditions. Plates were incubated at 37°C for 14 days. Cultures were then resuspended well, and OD₆₀₀ was determined using a Spectramax 5 (Molecular Devices).

Dragset M.S., Barczak A.K., Kannan N., Mærk M., Flo T.H., Valla S., Rubin E.J, & Steigedal M. (2015). Benzoic Acid-Inducible Gene Expression in Mycobacteria. PLoS ONE, 10(9), e0134544.

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