The formation of H2O2 from PMO activity in the absence of polysaccharide substrate was measured using a horseradish peroxidase (HRP)-coupled assay that oxidized Amplex red to produce a colorimetric readout. A PMO sample (2 µM) was incubated at RT with 100 µM Amplex red 1.3 µM HRP (Sigma), 2 mM ascorbate in 50 mM MOPS, pH 7.0. The absorbance at 540 nm was measured at 30 s intervals for 10 min on SpectraMax340 spectrophotometer (Molecular Devices, San Jose, CA). Each reaction was done in at least biological triplicate. Data are plotted with SEM error bars as calculated by GraphPad Prism 9.2.0. Data for HRP assays are provided in Figure 1—source data 3 , Figure 4—source data 4 , and Figure 5—source data 4 .
Spectramax 340 spectrophotometer
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax 340 is a spectrophotometer designed for accurate and reliable absorbance measurements. It features a wavelength range of 190 to 1,100 nm and can accommodate a variety of sample formats, including microplates, cuvettes, and test tubes. The SpectraMax 340 provides consistent and reproducible results, making it a versatile tool for a range of applications in life science research and development.
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Lab products found in correlation
Premix wst 1 cell proliferation assay system, by Takara Bio (2 mentions)
Prism 9, by GraphPad (1 mentions)
Gemcitabine, by LGC (1 mentions)
Celltiter 96, by Promega (1 mentions)
Penicillin streptomycin fungizone, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
6 protocols using spectramax 340 spectrophotometer
1
Peroxidase-Coupled Assay for H2O2 Production
2
Gemcitabine Resistance in Pancreatic Cancer
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Gemcitabine (GEM) was purchased from Toronto Research Chemicals, Inc. (Toronto, Ontario, Canada). First, we determined the half maximal inhibitory concentration (IC50) of GEM for MIA PaCa-2 or AsPC-1 cells using the Premix WST-1 Cell Proliferation Assay System (Takara Bio, Japan) according to the manufacturer’s instruction. Briefly, MIA PaCa-2 or AsPC-1 cells were seeded at a density of 2 × 103 cells per 100 μL in 96-well plates and allowed to adhere overnight. Then, cultures were re-fed with fresh media containing various concentrations of GEM. After 72 h of incubation, absorbance was measured at 450 nm in each well using a SpectraMax 340 spectrophotometer (Molecular Devices, CA, USA). The IC50 of GEM for each pancreatic cancer line was determined by constructing a dose-response curve. Each pancreatic cancer cell line was passaged in the cell lines’ IC50 concentration of GEM for 2 to 3 weeks. After passage, we again determined the cell lines’ IC50 value for GEM. Then, each pancreatic cancer cell line was passaged in the cell lines’ re-determined IC50 concentration of GEM for 2 to 3 weeks. The process was repeated at increasing doses of GEM until the cell lines demonstrated at least a 50-fold greater IC50 value for GEM than the parental cell lines. The resultant cell lines were resistant to GEM at a concentration of 20 μM.
3
Cytotoxicity Evaluation of SCU against PaCa Cell Lines
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The cytotoxicity of SCU against PaCa cell lines was evaluated using the Premix WST-1 Cell Proliferation Assay System (Takara Bio, Inc.) according to the manufacturer's protocol. MIA PaCa-2 and PANC-1 cell lines were seeded in 96-well plates at 2×103 cells/100 µl/well and cultured for 1 day. Various concentrations of SCU (1-200 µM) were then added to the cells; after 72 h of incubation at 37°C, absorbance was measured at 450 nm using a spectraMax 340 spectrophotometer (Molecular Devices, LLC).
4
In vitro Imaging Probe Evaluation
5
Cytotoxicity Evaluation of Probes on Bovine Chondrocytes
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The cytotoxicity of the probe to cells was determined via MTT assay as described previously49 (link), 50 (link). Bovine chondrocytes were isolated from the patella-femoral grooves of calves using previously established protocols51 (link). Briefly, calf knee joints were harvested from a local abattoir less than 36 hours after slaughter. Articular cartilage for in vitro study was collected from the patella-femoral grooves of only one donor, minced to approximately 1 mm3 pieces, and digested with 2 mg/mL collagenase type II (Worthington Biochemical Corp., Lakewood, NJ) in Dulbecco’s Modified Eagle’s Medium with 1% penicillin–streptomycin–fungizone (Life Technologies Corp., Carlsbad, CA) at 37 °C overnight. The chondrocyte solution was filtered through a 70 μm cell strainer, centrifuged, and then cultured in Dulbecco’s Modified Eagle’s Medium (DMEM). Six thousand cells were plated into each well of a 96-well plate and incubated overnight (37 °C), and then probes at various concentrations were added to the plate. After 24 hours of probe incubation (0 to 0.5 mg/ml), the MTT assay was carried out with the use of a SpectraMax 340 spectrophotometer (Molecular Devices, Sunnyvale, CA).
6
Relative Fitness of Silver-Resistant Bacteria
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Relative changes in the fitness of bacterial strains were determined in propagation assays. All strains were tested three times and the results were averaged. In vitro planktonic growth rates were measured for the parent strains and silver-resistant derivatives in monocultures with three different concentrations of silver nitrate (0, 5, and 315 μM Ag + ). Briefly, we adjusted the density of each bacterial suspension in brain-heart infusion broth (BHI; National Public Health Institute, Budapest, Hungary) supplemented by corresponding Ag + ion content to 0.5 McFarland (approximately 10 8 CFU/ml) using a McFarland turbidity meter. The working solutions were obtained after a 100-fold dilution. An amount of 200 μl of working solutions were pipetted into microtiter plates. Cultures were incubated at 35 °C with continuous shaking (260 rpm). Bacterial growth was measured after every 45 min by recording the absorbance at 620 nm using a SpectraMax 340 spectrophotometer (Molecular Devices, Sunnyvale, CA). Areas under curve (AUC) were computed to compare the relative fitness of the isolates.
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