Xmark microplate absorbance spectrophotometer

Manufactured by Bio-Rad

Sourced in United States, Japan, Canada, India, Italy

The XMark™ Microplate Absorbance Spectrophotometer is a laboratory instrument designed to measure the absorbance of light in microplates. It is capable of performing spectrophotometric analysis across a range of wavelengths.

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143 protocols using xmark microplate absorbance spectrophotometer

Pellicin Effects on K. xylinus Growth

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In order to determine the effect of pellicin on the growth of K. xylinus wild.
type and the plr15 mutant, cultures were examined in SH medium containing or lacking 10 μM pellicin. Growth kinetic data was collected from cultures grown in 96-well microtitre plates inoculated with a total volume of 200 μL SH medium containing 0.3% (v/v) cellulase and either DMSO or pellicin dissolved in DMSO. The inoculum was prepared by harvesting 5-day old, cellulase-digested cultures by centrifugation at 17,000 x g for 5 min at room temperature, followed by three washes with fresh SH medium. The starting inoculum was adjusted to an optical density at 600 nm (OD600) of 0.02 in SH broth. The bacterial culture plates were incubated at 30 °C with shaking at 150 rpm. The optical density was measured using Bio-Rad xMark™ Microplate Absorbance Spectrophotometer (Bio-Rad Laboratories Ltd., Mississauga, ON). Bacterial growth was monitored for 154 h. The data from eight technical replicates obtained from two biological replicates were averaged and used for statistical analysis.

Salgado L., Blank S., Esfahani R.A., Strap J.L, & Bonetta D. (2019). Missense mutations in a transmembrane domain of the Komagataeibacter xylinus BcsA lead to changes in cellulose synthesis. BMC Microbiology, 19, 216.

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Identification of Id2 Interactors and Tubulin Polymerization Assay

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HEK293T cells were transfected with GST–Id2, harvested, and lysed in low salt buffer (20 mM Tris-HCl pH 6.8, 1 mM MgCl₂, 2 mM EGTA, 0.5% NP-40, 1× protease inhibitor cocktail). Lysates were then centrifuged at 25,000 × g for 30 min at room temperature, and the supernatant was separated from the pellet. The pellet was resuspended in low salt buffer and sonicated. Equal volumes of supernatant and pellet samples were separated by SDS-PAGE and analyzed by immunoblotting. Tubulin polymerization assays were conducted using the CytoDYNAMIX Screen 03 assay system (Cytoskeleton, Inc.) following the manufacturer’s instructions. Tubulin (>99% pure) was reconstituted to 3 mg/mL in G-PEM buffer containing 80 mM PIPES, 2 mM MgCl₂, 0.5 mM EGTA, 1 mM GTP (pH 6.9), and 15% glycerol in the absence or presence of the indicated compounds at 4 °C. The mixture was added to each well of a prewarmed 96-well plate and exposed to test compounds at varying concentrations (0.1–10 μM/L). The absorbance at 340 nm was recorded every 60 s for 1 h at 37 °C using a Bio-Rad xMark Microplate Absorbance Spectrophotometer (Bio-Rad, Hercules, CA). Dose–response curves were plotted using Prism 7 (Graphpad Software, Inc.).

Yun T., Ko H.R., Jo D.G., Park K.W., Cho S.W., Kim J, & Ahn J.Y. (2021). Inhibitor of DNA binding 2 (Id2) mediates microtubule polymerization in the brain by regulating αK40 acetylation of α-tubulin. Cell Death Discovery, 7, 257.

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Cell Viability and Wound-Healing Assay

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Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Suit2mock and Suit2-STMN1 cells were plated at 5,000 cells per 100 μl medium in 96-well plates. Evaluations were performed at 48 h. To determine cell viability, 10 μl of cell counting solution was added to each well and the plates further incubated at 37˚C for 2 h. The absorbance of each well was then determined at 450 nm using a xMark Microplate Absorbance Spectrophotometer (Bio-Rad, Hercules, CA, USA).
Wound-healing assay. Suit2-mock and Suit2-STMN1 cells were plated in 6-well plates until confluence, and a uniform straight wound was produced in the monolayer in each well using a pipette tip. The wells were washed with PBS to remove all the cell debris, and the cells were cultured in 5% CO 2 at 37˚C. The relative closure rate of the wound was quantitatively evaluated at 72 h using brightfield microscopy.
Statistical analysis. Differences between groups were estimated using Student's t-test, chi-square analysis and analysis of variance. A result was considered statistically significant when the relevant p-value was less than 0.05. All statistical analyses were performed usingJMP software (SAS Institute Inc., Cary, NC, USA).

Suzuki K., Watanabe A., Araki K., Yokobori T., Harimoto N., Gantumur D., Hagiwara K., Yamanaka T., Ishii N., Tsukagoshi M., Igarashi T., Kubo N., Gombodorj N., Nishiyama M., Hosouchi Y., Kuwano H, & Shirabe K. (2018). High STMN1 Expression Is Associated with Tumor Differentiation and Metastasis in Clinical Patients with Pancreatic Cancer. Anticancer research, 38(2).

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Magnolin Inhibits Cancer Cell Proliferation

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Cells were seeded (AsPC‐1, 1 × 10³ cells/well; BxPC‐3, 1 × 10³ cells/well; Capan‐1, 1 × 10³ cells/well; Capan‐2, 1 × 10³ cells/well; MIA PaCa‐2, 1 × 10³ cells/well; SW480, 1 × 10³ cells/well; HCT 116, 1 × 10³ cells/well; HT‐29, 3 × 10³ cells/well; COLO 205, 1 × 10³ cells/well; MCF7, 1 × 10³ cells/well; MDA‐MB‐231, 1 × 10³ cells/well; BT‐474, 1 × 10³ cells/well; SKBR3, 1 × 10³ cells/well; TOV‐112D, 2 × 10³ cells/well; SKOV3, 1 × 10³ cells/well) into 96‐well plates in 100 µL of complete medium and incubated for 24 h; then, treated with vehicle (DMSO) or 15, 30, or 60 µM of magnolin for 24, 48, or 72 h. To the cells in each well were added 20 µL of the MTS‐based CellTiter 96^®Aqueous One Solution. The mixture was incubated for 1 h at 37°C in a 5% CO₂ incubator and then absorbance at 492 nm was measured by using an xMark™ Microplate Absorbance Spectrophotometer (Bio‐Rad Laboratories). Inhibition of cell proliferation by magnolin was evaluated by comparison with the absorbance of a vehicle‐treated control group over 72 h at 24 h intervals.

Song J., Lee C., An H., Yoo S., Kang H.C., Lee J.Y., Kim K.D., Kim D.J., Lee H.S, & Cho Y. (2018). Magnolin targeting of ERK1/2 inhibits cell proliferation and colony growth by induction of cellular senescence in ovarian cancer cells. Molecular Carcinogenesis, 58(1), 88-101.

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Mouse Brain Protein Extraction Protocol

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AD mouse brains were removed from mice 24 h after the last treatment of Ab‐TP‐MDNPs. The whole brain was homogenized in 1 mL of 1X radio‐immunoprecipitation assay buffer (RIPA) buffer (1X, pH 8.0) per 40 mg of brain tissue using a homogenizer (Fisher Scientific High Viscosity Homogenizer PowerGen 1000 S1, Waltham, MA). The homogenate was supplemented with 100 µL of 1X protease inhibitor mixture, and 200 µL each of 1X phosphatase inhibitor mixtures 1 and 2 (Sigma‐Aldrich, St. Louis, MO) per 10 mL of ice‐cold buffer and centrifuged at 14 000 × g for 10 min. The supernatant was then collected and used to measure protein content by ELISA as per the manufacturer's protocols (Bio‐Rad xMark Microplate Absorbance Spectrophotometer, Hercules, CA).

Park E., Li L.Y., He C., Abbasi A.Z., Ahmed T., Foltz W.D., O'Flaherty R., Zain M., Bonin R.P., Rauth A.M., Fraser P.E., Henderson J.T, & Wu X.Y. (2023). Brain‐Penetrating and Disease Site‐Targeting Manganese Dioxide‐Polymer‐Lipid Hybrid Nanoparticles Remodel Microenvironment of Alzheimer's Disease by Regulating Multiple Pathological Pathways. Advanced Science, 10(12), 2207238.

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MTT Proliferation Assay for Cell Cultures

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A 3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyl tetrazolium (MTT) assay was used to measure cell proliferation. The different cells were plated in 96-well plates and cultured for 1–4 days following transfection. Subsequently, 20 μl of MTT solution (5 mg/ml) were added to each well at the 12, 24, 48, 72 and 96 h time points. The absorbance at 570 nm was measured using a microplate reader (xMark™ Microplate Absorbance Spectrophotometer; Bio-Rad, CA, USA).

Li Y., Jiang D., Zhang Q., Liu X, & Cai Z. (2016). Ubiquitin-specific protease 4 inhibits breast cancer cell growth through the upregulation of PDCD4. International Journal of Molecular Medicine, 38(3), 803-811.

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Serological Diagnosis of Chagas Disease

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Sera samples were monitored for T. cruzi-specific IgG antibody response by an enzyme-linked immunosorbent assay (ELISA) by using a Wiener Chagatest-ELISA recombinant v.4.0 kit. The kit detects antibody response to six recombinant proteins that are expressed in T. cruzi. The assay was carried out following the manufacturer's recommendations, except that 2^nd antibody was replaced with goat anti-guinea pig IgG conjugated with HRP (sc2903, Santa Cruz Biotechnology, Dallas TX). Briefly, 96-well plates were coated with recombinant proteins, and then sequentially incubated with 20-μl sera samples (1:100 dilution) and HRP-conjugated guinea pig anti-IgG (1: 5000 dilution) diluted in phosphate buffer (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, and 1.4 mM KH₂PO₄, pH 7.4). The color was developed with tetramethylbenzidine and hydrogen peroxide substrates, and reaction was stopped by acidification of the reaction medium. The optical density was recorded at 450 nm by using an xMark microplate absorbance spectrophotometer (Bio-rad, Hercules, CA). The cut-off was determined from the mean value of the negative control sera samples ± 3 standard deviations. The sensitivity and specificity of the test was recorded at 99% and 98.3%, respectively.

Torres-Vargas J., Jiménez-Coello M., Guzmán-Marín E., Acosta-Viana K.Y., Yadon Z.E., Gutiérrez-Blanco E., Guillermo-Cordero J.L., Garg N.J, & Ortega-Pacheco A. (2018). Quantitative and histological assessment of maternal-fetal transmission of Trypanosoma cruzi in guinea pigs: An experimental model of congenital Chagas disease. PLoS Neglected Tropical Diseases, 12(1), e0006222.

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Quantification of Mouse IL-6 by ELISA

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ELISA was carried out per manufacturer’s instructions (R&D Systems, Quantikine™ ELISA Mouse IL-6, Catalog number M6000B, Minneapolis, MN). Reagent, samples, and standard solutions were prepared as directed and allowed to come to room temperature before use. A standard curve with known IL-6 concentrations (7.8–500 pg/mL) was run in parallel. Data from the technical replicate wells were averaged to obtain a mean value per plasma rich protein sample diluted at 1:2 before use. A positive control of mouse IL-6 sample of known concentration was also added. Plates were manually washed and aspirated between steps to lower background noise. Once the plate was complete it was read at a wavelength of 450 nm using Bio-Rad xMark™ Microplate Absorbance Spectrophotometer, with a correction wavelength of 540 nm.

Rashid M.H., Sparrow N.A., Anwar F., Guidry G., Covarrubias A.E., Pang H., Bogguri C., Karumanchi S.A, & Lahiri S. (2021). Interleukin-6 mediates delirium-like phenotypes in a murine model of urinary tract infection. Journal of Neuroinflammation, 18, 247.

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Quantitative Endotoxin Detection in Plasma

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LPS (endotoxin lipopolysaccharide) quantification was carried out per manufacturer’s instructions (Thermoscientific, Pierce™ Chromogenic Endotoxin Quant Kit, Catalog number A39553, Waltham, MA). All reagents were equilibrated to room temperature before use. Great care was taken to use materials that are endotoxin free. A standard of known endotoxin concentrations (0.100–0.010 EU/mL) was run in parallel with samples. Plasma samples were diluted at 1:50 in endotoxin free water supplied by the kit, and then heat shocked at 70 °C for 15 min before use. For the duration of this test the 96 wells plate was kept at 37 °C (Corning LSE Digital Dry Bath). Following colorimetric change wells were read at a wavelength of 405 nm using Bio-Rad xMark™ Microplate Absorbance Spectrophotometer.

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Cell Proliferation Assay Protocol

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After corresponding treatment, cells were suspended and counted using an
automated cell counter (Countess™ II Automated Cell Counter; Invitrogen,
Carlsbad, CA, USA). Approximately 3.0 × 10³ cells per well were
seeded into 96-well plates in triplicate. Then, cell viability was examined
using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto,
Japan) according to manufacturer’s protocol. Briefly, 10 μl of CCK-8 solution
was added to each well at the time-points of 0, 24, 48, 72, and 96 h. After 2 h
of culture at 37 °C, the optical density (OD) value was monitored with a plate
reader at 450 nm (xMark™ Microplate Absorbance Spectrophotometer; Bio-Rad,
Hercules, CA, USA). The cell growth curves were drawn based on OD values every
24 h.

Wang K, & Zhu Y. (2017). Dexmedetomidine protects against oxygen-glucose deprivation/reoxygenation injury-induced apoptosis via the p38 MAPK/ERK signalling pathway. The Journal of International Medical Research, 46(2), 675-686.

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