Spectramax m2e microplate reader

Manufactured by Molecular Devices

Sourced in United States

The SpectraMax M2e Microplate Reader is a versatile lab equipment designed for performing absorbance, fluorescence, and luminescence measurements on microplates. It provides a range of detection modes and capabilities to support various assays and applications in a laboratory setting.

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SpectraMax M2e Multi-Mode Microplate Reader (24 citations) SpectraMax M2/M2e Microplate Reader (18 citations) M2e microplate reader (3 citations) SpectraMax microplate reader (255 citations) SpectraMax M2e (642 citations) Microplate reader (2507 citations)

98 protocols using spectramax m2e microplate reader

Quantifying H2O2 in VSMC

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H₂O₂ was quantified using a kit (Amplex® Red Hydrogen Peroxide/Peroxidase Assay Kit, Molecular Probes), following the protocol provided by the manufacturer. Medium or serum samples, which were diluted 1:10 in reaction buffer, and standards were mixed with the working solution and incubated at room temperature for 30 min, protected from light. Fluorescence was excited at 535 nm and emission detected at 590 nm (Spectramax M2e Microplate Reader; Molecular Devices, Sunnyvale, CA). All samples were tested in duplicate and averaged.
To quantify the effect of catalase knockdown on cell content of H₂O₂, 48 h after transfection using either siRNA that targeted catalase or nontargeting siRNA, VSMC were then incubated in serum-free medium that contained 0 mM or 0.4 mM H₂O₂ for 5, 10, 15, 20, and 30 min. At each time point, medium was removed and cells were washed with PBS, then incubated with Amplex® Red reaction mixture (150 μl/well, 0.1 U horseradish peroxidase in 1x reaction buffer) for 1 h. Fifty μl of the reaction mixture was collected for each assay and fluorescence determined using an excitation wavelength of 535 nm and emission detected at 590 nm (Spectramax M2e Microplate Reader; Molecular Devices, Sunnyvale, CA). Live cells were counted from the same well with a Scepter Handheld Automated cell Counter (Cat#, PHCC00000, Millipore Corp. Billerica, MA).

Ying K.E., Feng W., Ying W.Z., Li X., Xing D., Sun Y., Chen Y, & Sanders P.W. (2022). Dietary salt initiates redox signaling between endothelium and vascular smooth muscle through NADPH oxidase 4. Redox Biology, 52, 102296.

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Quantifying PDGF-BB Expression in Engineered MSCs

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Unmodified MSCs, CMV-PDGF-BB-MSCs, and PGK-PDGF-BB-MSCs were cultured in GM and seeded at a density of 2 × 10⁴ cells/well onto 24-well plates. The samples, which included the supernatant and cells, were collected at one, three, and seven days. For the samples collected at seven days, GM was replaced with fresh GM at three days. After collecting the supernatants, each plate was then washed three times with PBS and 1 mL of DNase/RNase free water was subsequently added into each well. Cellular DNA was released by three freeze–thaw cycles and the quantity of dsDNA in each well was measured by performing a Quant-iT PicoGreen dsDNA Assay (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Fluorescence was measured at 480/520 nm wavelength using a plate reader (SpectraMax M2e Microplate Reader, Molecular Devices, San Jose, CA), and the amount of dsDNA was calculated.
To verify expression of PDGF-BB, the supernatant at three and seven days was analyzed. The concentration of PDGF-BB was measured using a human PDGF-BB ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions. Optical absorbance at 450 nm was measured using a plate reader and PDGF-BB expression level was calculated.

Guzman R.A., Maruyama M., Moeinzadeh S., Lui E., Zhang N., Storaci H.W., Tam K., Huang E.E., Utsunomiya T., Rhee C., Gao Q., Yao Z., Yang Y.P, & Goodman S.B. (2021). The effect of genetically modified platelet-derived growth factor-BB over-expressing mesenchymal stromal cells during core decompression for steroid-associated osteonecrosis of the femoral head in rabbits. Stem Cell Research & Therapy, 12, 503.

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Quantitative real-time RT-PCR analysis of gene expression

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Total RNA was extracted from mycelia transferred to medium containing glucose or no carbon source for 16 hr as described previously (Reinert et al. 1981 (link)) and treated with the Ambion Turbo DNA-free Kit (Invitrogen, AM1907, Carlsbad, CA) prior to quantification in a SpectraMax M2e Microplate Reader (Molecular Devices, M2E, Sunnyvale, CA). The primers used in qRT-PCR experiments were designed using the Primer3 program (http://frodo.wi.mit.edu/primer3/) and are listed in Table S1. Each primer pair was first tested with serial dilutions of RNA to determine the linear range of the qRT-PCR assays using the SuperScript III Platinum SYBR Green One-Step qRT-PCR Kits (Invitrogen, 11736). The experiments were performed using a Corbett CAS1200 liquid handling robot and Corbett Rotor-Gene 3000 real-time thermal cycler (QIAGEN, RG3000, Hilden, Germany). In the assays to determine relative transcript levels, 1 ng of total RNA was added to each reaction. A minimum of three independent RNA preparations were assayed.

Katz M.E, & Cooper S. (2015). Extreme Diversity in the Regulation of Ndt80-Like Transcription Factors in Fungi. G3: Genes|Genomes|Genetics, 5(12), 2783-2792.

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Quantifying GFP Fluorescence in Bacteria

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To quantify the green fluorescence intensity of the GFP, 1 ml of GFP expressing bacterial cells were harvested, washed and re-suspended in 500 μl 1X PBS (phosphate buffered saline). Fluorescence intensities of the re-suspended cells were measured in a black opaque 96 well plate (Corning^®, USA) at excitation of 490 nm and emission of 510 nm using the SpectraMax M2e microplate reader (Molecular Device, USA). Fluorescence values were normalized with optical density of the bacterial cells.

Srivastava S.K., Iyer V.R., Ghosh T., Lambadi P.R., Pathania R, & Navani N.K. (2016). Isolation of a non-genomic origin fluoroquinolone responsive regulatory element using a combinatorial bioengineering approach. Nucleic Acids Research, 44(5), 2451-2461.

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Mitochondrial Respiration Assay in RCC Cells

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RCC4 or RCC10 cells ectopically expressing vector or FBP1 were seeded into black opaque 96-well plate at a density of 70, 000 cells per well. After attachment, culture medium within each well was replaced with 150 µL oxygenated fresh medium, supplemented with 10 µL MitoXpess probe solution (Cayman Chemical). Antimycin A was used as negative control. 100 µL mineral oil was dispensed to overlay each well, and the whole plate was monitored using SpectraMax m2e microplate reader (Molecular Devices) at 37 °C. Wavelengths are set to 380 nm for excitation and 650 nm for emission.

Li B., Qiu B., Lee D.S., Walton Z.E., Ochocki J.D., Mathew L.K., Mancuso A., Gade T.P., Keith B., Nissim I, & Simon M.C. (2014). Fructose-1, 6-bisphosphatase opposes renal carcinoma progression. Nature, 513(7517), 251-255.

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Effect of IL-4 Preconditioning on MSC Proliferation

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The experimental outline is shown in Figure S1. MSCs or IL4-MSCs were seeded in T75 flasks and cultured with MSC growth medium for 1 day. For the MSC and IL4-MSC groups, the medium was replaced by fresh MSC growth medium and cells were cultured for 3 days. For the pMSC and IL4-pMSC groups, the cells were cultured with the MSC preconditioning medium (MSC growth medium containing TNFα [20 ng/mL] and LPS [20 μg/mL]) for 3 days. The cells in all four groups were washed with Dulbecco’s phosphate-buffered saline (DPBS) three times, then the cells were trypsinized and seeded onto a 6-well plate (2 × 10⁴ cell/well) with the MSC growth medium. The samples including the supernatants and cells were collected at day 1, 3, and 7. For the samples at day 7, the medium was replaced with fresh medium at day 3. The supernatants were stored at −80 °C and were used for the other experiments.
Cell proliferation was analyzed using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA). After washing three times with DPBS, 2 mL of DNase/RNase free water was added into each well. Cellular DNA was released by three freeze-thaw cycles and stained with PicoGreen according to the manufacturer’s protocol. Fluorescence was measured at 480/520 nm wavelength using a plate reader (SpectraMax M2e Microplate Reader; Molecular Devices, San Jose, CA), and the amount of dsDNA was calculated.

Maruyama M., Moeinzadeh S., Guzman R.A., Zhang N., Storaci H.W., Utsunomiya T., Lui E., Huang E.E., Rhee C., Gao Q., Yao Z., Takagi M., Yang Y.P, & Goodman S.B. (2021). The efficacy of lapine preconditioned or genetically modified IL4 over-expressing bone marrow-derived mesenchymal stromal cells in corticosteroid-associated osteonecrosis of the femoral head in rabbits. Biomaterials, 275, 120972.

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Heterologous Expression and Purification of Ghlac

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The gene encoding Ghlac WT (NCBI accession No.: ORJ60343.1, https://www.ncbi.nlm.nih.gov/protein/ORJ60343.1, accessed on 15 June 2019) was codon-optimized, synthesized, and cloned in the pET-28a (+) vector using the Nco I and Xho I restriction sites. The obtained recombinant vector pET28a-Ghlac-WT was transformed into E. coli BL21 (DE3). The cells containing the recombinant vector were grown in Luria–Bertani (LB) medium supplemented with 25 mg/L of kanamycin. When OD₆₀₀ reached 0.6, 0.5 mM IPTG and 0.5 mM CuSO₄ were added to induce the expression of Ghlac, and then the cells continued to grow at 16 °C for 16 h. The cells were collected by centrifugation at 4000× g for 30 min and homogenized using a JN-Mini homogenizer (JNBio, Guangzhou, China). The recombinant Ghlac in the supernatant was purified using Ni-NTA resin according to the reported method [50 (link)]. The purified Ghlac in 20 mM phosphate buffer (pH 7.4) was stored at −80 °C. The purity and molecular mass of Ghlac were assessed by SDS-PAGE. The UV/visible absorption spectrum of Ghlac was scanned in the range of 200–800 nm using a SpectraMax M2e Microplate Reader (Molecular Devices, Sunnyvale, CA, USA). The copper content of Ghlac was analyzed with an iCAP Qc inductively coupled plasma mass spectrometry (ICP-MS) (ThermoFisher Scientific, Waltham, MA, USA) [22 (link)].

Mao G., Wang K., Wang F., Li H., Zhang H., Xie H., Wang Z., Wang F, & Song A. (2021). An Engineered Thermostable Laccase with Great Ability to Decolorize and Detoxify Malachite Green. International Journal of Molecular Sciences, 22(21), 11755.

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Quantifying DiI-LDL Uptake in HepG2 Cells

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DiI-LDL uptake assays were conducted as described previously [21 (link)], with minor modifications. In brief, HepG2 cells were seeded in 24-well plates. After specific treatments, the culture medium was changed to DiI-LDL DMEM (20 μg/mL), and the cells were incubated for 3 h at 37 °C in the dark. Then, the cells were rinsed twice with ice-cold PBS buffer containing 0.4% albumin (Sigma-Aldrich) and washed twice with ice-cold PBS buffer. Then, 500 μL of isopropanol was added into each well, and the cells were incubated for 20 min in the dark at room temperature with constant shaking. Aliquots of 200 μL were used for fluorescence detection with the SpectraMax M2e Microplate Reader (520–578 nm, Molecular Devices, San Jose, CA, USA).

Li H.H., Li J., Zhang X.J., Li J.M., Xi C., Wang W.Q., Lu Y.L, & Xuan L.J. (2019). 23,24-Dihydrocucurbitacin B promotes lipid clearance by dual transcriptional regulation of LDLR and PCSK9. Acta Pharmacologica Sinica, 41(3), 327-335.

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Osteoblastic Differentiation Assay using Fucoidan

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MC3T3-E1 osteoblastic cells were purchased from the American Type Culture Collection (USA) and cultured in α-MEM containing 10% FBS, 100 μg/ml streptomycin, and 100 U/ml penicillin on the polystyrene-coated dishes. Cell densities were maintained so as not to exceed 70-80% confluency. Briefly, MC3T3-E1 cells were seeded into a 96-well plate at a density of 1 × 10⁴ cells/well and cultures for 24 h. And α-MEM-based medium was replaced with fresh differentiation medium containing 10% FBS, 1% penicillin-streptomycin, 10 mM β-glycerophosphate and 100 mg/ml ascorbic acid as osteogenic agents. And the cells were treated with ultrafiltrated-fucoidan fractions at different concentrations and incubated for an additional 7 days. To determine ALP activity using a colorimetric assay, cells were washed with 1x PBS and lysed by incubation in 100 μl of lysis buffer (0.5 M Tris, pH 9.0, 150 mM NaCl, 1% Triton X-100) for 1 h at 4°C. Cell lysates were then treated with 100 μl of 5 mM p-nitrophenyl phosphate solution for 30 min at 37°C and 50 μl of resulting supernatants were transferred to new 96-well plates. The reaction was stopped by adding an equal volume of 0.1 N NaOH and absorbances were measured at 405 nm using a SpectraMax M2e microplate reader (Molecular Devices). All data were expressed as percentages of non-treated group.

Park B., Yu S.N., Kim S.H., Lee J., Choi S.J., Chang J.H., Yang E.J., Kim K.Y, & Ahn S.C. (2022). Inhibitory Effect of Biotransformed-Fucoidan on the Differentiation of Osteoclasts Induced by Receptor for Activation of Nuclear Factor-κB Ligand. Journal of Microbiology and Biotechnology, 32(8), 1017-1025.

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QD-based Fluorescence Immunoassay Protocol

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The procedures for FLISA and ic-ELISA were identical before the secondary antibody was added. More specifically, after washing with PBST for three times, 100 μL of 655 nm QDs-conjugated goat anti-mouse IgG (1:10,000 dilution) was added to each well and incubated at 37 °C for 45 min. After three washes with PBST, a SpectraMax M2e Microplate Reader (Molecular Devices, San Jose, CA, USA) was used to measure the fluorescence signals from samples with excitation/emission at 360/655 nm. The fluorescence intensities were identified as B₀ and B for the control and testing sample. The standard curve was established by plotting the form (B/B₀) 100% against the 2-NP-AMOZ concentration.

Xie Y., Wang Y., Yan X., Gan L, & Le T. (2021). Preparation of Monoclonal Antibody and Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay and Fluorescence-Linked Immunosorbent Assay for Detecting 3-Amino-5-methylmorpholino-2-oxazolidinone (AMOZ) in Edible Animal Tissue. Molecules, 26(14), 4243.

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