WST-1 reagent (cat. no. 11644807001; Roche Diagnostics GmbH) was used to detect cell proliferation. Transfected cells were plated in 96-well culture plates at a density of 3×103 cells/well. After 1, 2, and 3 days, cells were incubated with 10% WST-1 solution for 2 h at 37°C. The absorbance was measured at 450 nm using an Infinite M200 spectrophotometer (Tecan Group, Ltd.). Three replicates of each experiment were carried out.
Infinite m200 spectrophotometer
Manufactured by Tecan
Sourced in Switzerland, Austria, United States
The Infinite M200 spectrophotometer is a high-performance instrument designed for precise and reliable absorbance measurements. It features a wide wavelength range, adjustable bandwidth, and advanced optics to ensure accurate and reproducible results.
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Lab products found in correlation
Annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (4 mentions)
Cell counting kit 8 cck 8, by Dojindo Laboratories (4 mentions)
Cell counting kit 8 (cck8), by Dojindo Laboratories (3 mentions)
Fluorometric assay kit, by Abcam (3 mentions)
Cell light edu apollo 567 in vitro imaging kit, by RiboBio (3 mentions)
Fluorescence microscope, by Olympus (3 mentions)
Human fibrinogen, by Enzyme Research (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ldh cytotoxicity colorimetric assay kit, by Abcam (2 mentions)
Cell proliferation kit 2 xtt, by Roche (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Rna 6000 nano labchip kit, by Agilent Technologies (2 mentions)
Mts reagent, by Promega (2 mentions)
Vybrant mtt cell proliferation assay kit, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Fibrinogen, by Corning (1 mentions)
Fibrinogen, by Merck Group (1 mentions)
Thrombin, by Merck Group (1 mentions)
105 protocols using infinite m200 spectrophotometer
1
Cell Proliferation Assay using WST-1
2
Fibrinogen-based Composite Scaffold Fabrication
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fibrinogen was prepared as described previously (Moreira et al., 2016 (link)). In short, dissolved lyophilized fibrinogen (Calbiochem) was dialyzed against tris-buffered saline (TBS; pH 7.4) for 12 h utilizing a 6000–8000 molecular weight cut-off membrane (Novodirect). The obtained fibrinogen solution was sterilized by filtration (0.2 μm pore size, Corning®), and the concentration was defined by measuring the absorbance at 280 nm using an Infinite M200 spectrophotometer (Tecan Group Ltd.). The fibrin gel components of MEW/fibrin composite (5.0 mL in total) consisted of 2.5 mL fibrinogen solution (10 mg/mL), and the fibrin polymerization starting solution composed of 1.75 mL TBS, 0.375 mL 50 mM CaCl-2 (Sigma) in TBS, and 0.375 mL 40 U/mL thrombin (Sigma). The components were introduced into a 3D printed (Objet Eden350) square shaped mold of 225 mm2 to composite the scaffolds for burst strength tests.
3
Ellman Principle-Based AChE Activity Assay
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The enzyme activity assay is based on the Ellman principle, as described previously (Ellman et al. 1961 (link); Worek et al. 1999 (link)). Briefly, 20 µL of electric eel AChE or 20 µL of Triton-diluted blood (either exposed or unexposed) and 20 µL ethopropazine were added to 460 µL of DTNB buffer in a 48-wells plate. The plate was incubated for 20 min at room temperature to allow for completion of the reaction between blood matrix thiols and DTNB, and to allow for the complete inhibition of BChE by ethopropazine. After incubation, 100 µL of acetylthiocholine was added and absorbance was read at 436 nm for 10 min using a Tecan infinite M200 spectrophotometer (Tecan Group, Ltd., Männedorf, Switzerland). Blank wells contained only DTNB buffer and unexposed enzyme/blood to correct for any background absorbance. To these wells, 100 µL sodium phosphate buffer was added instead of acetylthiocholine after incubation. The final concentration of electric eel AChE was 4 mU/well.
4
Sulforhodamine B Colorimetric Assay
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After the treatment time specified in the experimental conditions, cells were fixed with trichloroacetic acid (TCA; Sigma-Aldrich, St. Luis, MO, USA) at a final concentration of 12.5% and incubated for 1 h at 4°C, followed by gentle rinsing with cold water and drying. Subsequently, a freshly prepared solution of 0.04% SRB (Sigma-Aldrich, St. Luis, MO, USA) in 1% acetic acid (Avantor Performance Materials Poland, Gliwice, Poland) was added and left at room temperature for 30 min. The unbound dye was removed using 1% acetic acid. The protein-bound SRB was solubilized in 10 mM Tris base solution (BioShop, Burlington, Ontario, Canada), pH 10.5. The absorbance, proportional to the protein content, was measured using the Infinite® M200 spectrophotometer (Tecan Group Ltd., Mannedorf, Switzerland) at λ = 520 nm. The experiments were performed in triplicate, utilizing cells from different cell passages.
5
MTT Cell Viability Assay Protocol
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After the treatment time specified in the experiment conditions, the post-culture medium was discarded, cells were rinsed with sterile phosphate-buffered saline (PBS; Gibco, Thermo Fisher Scientific, Waltham, MA, USA) solution, and a freshly prepared 0.5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide in complete growth medium (MTT reagent; Sigma-Aldrich, St. Luis, MO, USA) was added to the culture. Plates were incubated for 3 h in the CO2 incubator under the above-mentioned conditions. Subsequently, the MTT reagent was gently decanted, and the formed formazan crystals were dissolved in DMSO (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The absorbance was measured with Infinite® M200 spectrophotometer (Tecan Group Ltd., Mannedorf, Switzerland) at λ = 540 nm. The experiments were conducted in triplicate, utilizing cells from different cell passages.
6
Fibroblast Collagen Production Assay
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Skin fibroblasts were seeded into T25 flask (5×105/T75 flask, n=6) or into scaffolds (5×105/scaffold, n=6) and grown in maintenance medium (2% FBS as control), 5% PRCR and 10% PRCR for 7 days. After the cell-conditioned medium was collected, cells were detached by trypsinisation. To measure total soluble collagen, total soluble collagen kit was used (QuickZyme BioSciences, Leiden-Netherlands). Briefly, the cells were trypsinised in both culture systems, washed with PBS and equal number of cells were used for this assay. The PBS was replace by 0.5 M Acetic acid (250 μl/well of 24-well-plate), incubate overnight at 4oC on a rotating platform, The cellular extract was centrifuged 10 minutes 3.000 x g, and the supernatant was tested in the assay in 1-fold to 10-fold dilutions made in dilution buffer. Measurements were performed at a wavelength of 540 nm in triplicates using Tecan Infinite M200 spectrophotometer (Tecan Group Ltd., Switzerland). A standard curve for calculating collagen concentration was obtained using a manufacturer-supplied acid soluble type I collagen calibration standard solution.
7
Colorimetric Assay for Muscle Glutamine Synthetase
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Glutamine synthetase activities in muscles were determined using a colorimetric assay based on the formation of γ-glutamyl-hydroxamate (Minet et al. 1997 ). Tissue samples were homogenized in imidazole buffer (pH 6.8, 0°C) with excess of GLN and hydroxylamine (1/9 w/v) and incubated at 37°C for 30 min. After centrifugation, the colour created by reaction with ferric chloride was measured at 530 nm in Tecan Infinite M200 spectrophotometer (Tecan Group Ltd., Switzerland). The results were expressed as relative units obtained by normalizing of the glutamine synthetase activity for control of SOL (A SOL = 1).
8
Aptamer Enrichment from R. microfusus
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After pretreatment with R. microfusus (see Section 3.3.1 ), R. microfusus (1 mL OD600 = 0.1) was mixed with 5 pmol of Cy5-labeled activated aptamer library in 500 µL of 1 × DPBS and incubated at 37 °C for 30 min. The supernatant was removed after centrifugation at 3000× g for 2 min, washed once, and the pellet was resuspended with 100 µL of 1 × DPBS buffer to obtain the eluted aptamers combined with cells. Finally, fluorescence intensity was measured using an Infinite M200 spectrophotometer (TECAN Trading AG, Männedorf, Switzerland) at 635 nm excitation and 670 nm emission.
9
Biofloc System Water Quality Monitoring
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Temperature, dissolved oxygen, pH, total ammonia, nitrite and total suspended solids (TSS) were determined daily, whereas alkalinity, nitrate and volatile suspended solids (VSS) were analyzed once per week (Table 1). Total ammonia, considered a critical parameter in BFT systems, was measured in two ways: once a week by means of the indophenol method (Strickland and Parsons, 1972) using microplates and absorbance read at 240 nm (Infinite M200 spectrophotometer; Tecan Trading AG, Switzerland), and daily with a Merck colorimetric kit (MColortest™). Due to the water turbidity of the biofloc systems, all samples were diluted 1/10 to better visualize the colorimetric card. The values of the colorimetry test were correlated with those of the analytical method to generate an equation to correct the subjectivity of the colorimetric method. The values of non-ionized ammonium (NH 3 ) were calculated from the concentration of total ammonium (NH 4 + ), pH and temperature of water (Boyd, 1990) .
10
LDH Leakage and Cell Viability Assay
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Lactate dehydrogenase (LDH) leakage test consisted of measurement of LDH activity in the culture medium and in the cell lysate (hepatocytes were frozen for 10 min at -80°C and lysed in distilled water) using a commercial kit from DiaSys (Holzheim, Germany). The ratio of extracellular and total LDH was calculated.
Cell viability was assessed using the WST-1 assay (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl) -2H-tetrazolium, monosodium salt, Roche, Germany) evaluating the activity of intracellular dehydrogenases (described by Lotková et al. 2005 (link)). In the assay, the maternal tetrazolium salt WST-1 is cleaved to formazan by intracellular dehydrogenases. The higher the number of viable cell is, the higher the activity of the enzymes leading to the higher production of the formazan dye. The change directly correlates to the number of metabolically active cells in the culture. The medium was collected and the WST-1 reagent (diluted 1:10 in PBS; obtained from Roche, Penzberg, Germany) was added to the well-plates. Changes in absorbance of the dye solution were measured at time 0, 1 h and at 2 h using a TECAN Infinite M200 spectrophotometer (Tecan Group AG, Männedorf, Switzerland) at a wavelength of 440 nm. The difference between these values was calculated and used for statistical analysis.
Cell viability was assessed using the WST-1 assay (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl) -2H-tetrazolium, monosodium salt, Roche, Germany) evaluating the activity of intracellular dehydrogenases (described by Lotková et al. 2005 (link)). In the assay, the maternal tetrazolium salt WST-1 is cleaved to formazan by intracellular dehydrogenases. The higher the number of viable cell is, the higher the activity of the enzymes leading to the higher production of the formazan dye. The change directly correlates to the number of metabolically active cells in the culture. The medium was collected and the WST-1 reagent (diluted 1:10 in PBS; obtained from Roche, Penzberg, Germany) was added to the well-plates. Changes in absorbance of the dye solution were measured at time 0, 1 h and at 2 h using a TECAN Infinite M200 spectrophotometer (Tecan Group AG, Männedorf, Switzerland) at a wavelength of 440 nm. The difference between these values was calculated and used for statistical analysis.
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