PM proteins and vesicles were isolated from 7-dag Col-0 WT and Qabi2-2 roots according to Zhu et al. (82 (link)) with minor modifications. PM protein content was calculated using the Bradford assay. Ten micrograms of PM proteins were used to determine the AHA activities by measuring the release of Pi from ATP according to the method of Kinoshita and Shimazaki (43 (link)) using an ultraviolet (UV) spectrophotometer (Spectramax 250, Molecular Devices, Sunnyvale, CA, USA) under the reaction temperature 30°C. The H+ transport activity was measured as the quenching of A492 by acridine orange after adding a mixture of Mg-ATP (MgSO4 and disodium ATP) at a final concentration of 1 mM using a UV spectrophotometer (Spectramax 250, Molecular Devices, Sunnyvale, CA, USA) under the reaction temperature 30°C according to the method of Zhu et al. (82 (link)). To determine the PM purity, AHA activity was monitored in the amount of released Pi with or without specific inhibitors including sodium VA (0.02 mM; pH 6.5) for PM, nitrate (50 mM; pH 8) for tonoplast, and azide (1 mM; pH 8) for mitochondrial and endoplasmic membrane according to the method of Zhu et al. (82 (link)). Each experiment was repeated three times, independently.
Spectramax 250
Manufactured by Molecular Devices
Sourced in United States, Germany, Canada
The SpectraMax 250 is a multimode microplate reader designed for absorbance, fluorescence, and luminescence detection. It is capable of performing a variety of assays, including cell-based, biochemical, and molecular biology applications. The instrument features a monochromator-based optical system that provides flexible wavelength selection and optimization.
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Lab products found in correlation
Methyl thiazolyl tetrazolium (mtt), by Merck Group (11 mentions)
Xtt cell viability assay kit, by Sartorius (7 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (5 mentions)
Mtt reagent, by Merck Group (4 mentions)
Goat anti mouse igg igg1 igg2a igg2b igg3 antibodies labeled with horseradish peroxidase, by Southern Biotech (3 mentions)
Immuno plate, by Thermo Fisher Scientific (3 mentions)
Mts assay, by Promega (3 mentions)
Shla g kit, by Exbio (3 mentions)
Softmax pro software, by Molecular Devices (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Cyquant xtt cell viability assay, by Thermo Fisher Scientific (2 mentions)
Dhr 123, by Agilent Technologies (2 mentions)
Dhr123, by Merck Group (2 mentions)
A5533, by Merck Group (2 mentions)
Picrosirius red, by ScyTek Laboratories (2 mentions)
Picro sirius red, by Molecular Devices (2 mentions)
Bouin s fixative, by Electron Microscopy Sciences (2 mentions)
Picric acid, by Merck Group (2 mentions)
Xtt cell viability assay, by Sartorius (2 mentions)
Prism 5, by GraphPad (2 mentions)
249 protocols using spectramax 250
1
Isolation and Characterization of Plant Plasma Membrane Proteins
2
Cell Viability Assay with MTT
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For evaluation of cell survival, cells were seeded on 24-well plates at a density of 2 × 104 cells/well, followed by the addition of methyl thiazol tetrazolium (MTT; Sigma) at the end of cell culture. The amount of MTT formazan product was determined using a microplate reader at an absorbance of 560 nm (SpectraMax 250, Molecular Devices).
3
Evaluating Glioma Cell Viability
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The effect of tamoxifen and temozolomide on cell viability was assessed in glioma cell lines and primary cultures (G1) by MTT 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide assay. Cells were treated for 24 h with drugs or with DMSO (vehicle control). MTT (5 mg/ml) was added and formazan crystals formed were dissolved in 10% SDS along with 0.01 N HCl. The absorbance was measured at 570 nm with reference to 640 nm using microplate reader (Molecular Devices, SPECTRA max 250, USA) and percent viable cells was calculated considering values in control as 100%. Vehicle control cells showed cell viability >95%.
4
Cytotoxicity of Au NPs on MSCs
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Mesenchymal stem cells were seeded at a density of 104 cells/well into a 96-well plate with each well filled with Vmedium = 0.1 mL of medium and exposed to Au NPs for 24–72 h. Then, cells were washed once with PBS and AlamarBlue (Thermo Fisher Scientific) was added in each well and incubated for desired time at 37 °C. The fluorescence was measured at 560 nm excitation and 590 nm emission wavelengths using a spectrophotometer (SpectraMax 250, Molecular Devices). Cell viability was assumed to be proportional to the recorded fluorescence intensity. Results are expressed as percentage of cell viability V versus control (i.e. untreated cells). Experiments were performed with MSCs from three independent human/MSC donors in triplicates for each time-point and concentration.
5
Cell Proliferation Assay Using CCK-8
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We assessed cell proliferation using the Cell Counting Kit-8 (CCK-8) assay (Dojindo, Shanghai, China). In brief, we seeded cells (3 × 104) into plates with 96 wells and incubated them at 37 °C for 1 day before transfection. We added CCK-8 solution (10 μL) to every well 2 days after transfection. At the indicated time points, we measured absorbance at a wavelength of 450 nm for 5 days using a Spectra Max 250 spectrophotometer (Molecular Devices, San Jose, CA, USA). The experiments were performed in triplicate.
6
Extraction and Quantification of Liver Lipids
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Total lipids from the liver were extracted using the modified method of Folch, et al. (1957) . Briefly, 250 mg of frozen liver tissue from the same region of the liver was weighed and transferred into a 2-mL flat-bottom centrifuge tube containing 0.5 mL methanol. After homogenization, 0.5 mL of chloroform and 0.4 mL of dist. water were added to the liver homogenate and mixed by vortexing. The lipid fraction in chloroform was separated from the aqueous fraction and liver debris by centrifuging for 10 min at 14,000 rpm at 20°C and was then transferred to a new glass tube. After drying the lipid fraction was reconstituted in n-butanol for further analysis of TC. TC concentrations were determined enzymatically by conducting colorimetric assays (Pointe Scientific, Canton, MI) in a 96-well plate reader (SpectraMAX 250, Molecular Devices, Sunnyvale, CA).
7
Cytokine Quantification in Mouse Lung
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The quantification of cytokines in mouse lung was conducted by a commercial ELISA kit (R&D Systems) according to the manufacturer's instructions. The individual sample's optical density was measured at 450 nm using a spectrophotometer (Spectra Max-250, Molecular Devices, Sunnyvale, CA, USA).
8
Tyrosinase Activity Assay with L-dopa
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Tyrosinase activity using L-dopa as the substrate was assayed spectrophotometrically. A 25 μl sample of NDGA was added to the assay mixture containing 5 mM L-dopa solution, 50 mM phosphate buffer (pH 7.4) and 25 μl of 0.6 mg/ml mushroom tyrosinase solution which was added to a 96-well microplate for a total volume of 200 μl. The assay mixture was incubated at 37°C for 10 min. After incubation, the absorbance was measured at 475 nm in a model SPECTRAmax 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
9
Cell Proliferation Assessment by CCK-8 Assay
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Cell proliferation was assessed by Cell Counting Kit-8 assay (Dojindo Laboratories, Japan). Cells (1 × 103) were seeded into 96-well plates and incubated at 37 °C for 24 h before transfection. CCK-8 solution(10 μl) was added to each well 48 h after transfection. After 2 h of incubation at 37 °C, the absorbance at 450 nM was measured using Spectra Max 250 spectrophotometer (Molecular Devices, USA). Triplicate independent experiments were performed.
10
Measuring Antioxidant Capacity by DPPH Assay
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Radical scavenging activity of Mc-ME was determined by a DPPH assay as previously reported [29 (link)]. DPPH (150 μM) was mixed with either Mc-ME (0–200 μg/ml) or ascorbic acid (100 μM) and incubated for 20 min at room temperature. Absorbance was determined at 517 nm using a Spectra Max 250 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
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