For detection of antibodies in serum, plates were coated with rat-anti-mouse IgE (R35-72; BD) or goat-anti-mouse IgG (Southern Biotech) overnight at 4°C. Purified mouse IgE (BD) or unlabelled mouse IgG1, IgG2a or IgG2b (all Southern Biotech) were used to generate a standard curve. Goat-anti-mouse IgE-AP, IgG1-AP, IgG2a-AP or IgG2b-AP (all Southern Biotech) were used for detection. For detection of parasite-specific antibodies, plates were coated with 20 μg/mL of HES suspension overnight at 4°C. After blocking for 2 hrs with 3% BSA, samples were incubated at room temperature for 2 hrs and the AP-coupled antibodies named above were used for detection. Absorption was measured on a Multiscan FC photometer (Thermo Fisher) at 405 nm and blank wells were used to subtract background.
Multiscan fc photometer
Manufactured by Thermo Fisher Scientific
Sourced in Italy
The Multiscan FC photometer is a versatile instrument designed for absorbance-based measurements. It can be used for a variety of applications, including enzyme-linked immunosorbent assays (ELISAs), cell-based assays, and other colorimetric techniques. The Multiscan FC provides accurate and reliable data, with a wide range of wavelengths and adjustable measurement parameters to suit different experimental needs.
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Lab products found in correlation
Rat anti mouse ige r35 72, by BD (1 mentions)
Purified mouse ige, by BD (1 mentions)
Igg1 ap, by Southern Biotech (1 mentions)
Igg2b ap, by Southern Biotech (1 mentions)
Goat anti mouse igg, by Southern Biotech (1 mentions)
Glass beads, by Carl Roth (1 mentions)
F96 microwell plate, by Thermo Fisher Scientific (1 mentions)
Matlab r2015b, by MathWorks (1 mentions)
7 protocols using multiscan fc photometer
1
Quantification of Mouse Ig Isotypes
2
Evaluating Cytotoxicity with Microtetrazolium Test
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Microtetrazolium test [21 (link)] was used to study cytotoxicity of the compounds. Briefly, series of two-fold dilutions of each compound (300−4 μg/ mL) in MEM were prepared. MDCK cells (ATCC CCL-34) were incubated for 48 h at 37 °C in 5% CO2 in the presence of the dissolved substances. The degree of destruction of the cell monolayer was then evaluated in the microtetrazolium test (MTT). The cells were washed twice with saline, and a solution of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (ICN Biochemicals Inc., Aurora, Ohio) (0.5 mg/ mL) in phosphate-buffered saline was added to the wells. After 1 h incubation, the wells were washed and the formazan residue dissolved in DMSO (0.1 mL per well). The optical density of wells was then measured on a Multiscan FC photometer (Thermo Scientific, USA) at wavelength of 540 nm and plotted against concentration of the compounds. Each concentration was tested in three parallels. The 50 % cytotoxic dose (CC50) of each compound (i.e., the compound concentration that causes the death of 50 % cells in a culture, or decreasing the optical density twice as compared to the control wells) was calculated from the data obtained. Values of CC50 obtained in microgram/mL were then calculated into micromoles.
3
Enzymatic Assays in E. coli Lysates
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Enzyme activities were determined in cell lysates. Therefore, 1 mL E. coli cell suspension was mixed with 0.5 g glass beads (ø 0.25–0.5 mm, Carl Roth, Karlsruhe, Germany) and disrupted for 5 min and 25 s−1 in a Mixer Mill MM 400 (Retsch, Haan, Germany). Debris was separated by centrifugation (17,200× g, 10 min, 4 °C) and the activity in the supernatant was determined photometrically by oxidation (ER) or reduction (FDH) of 500 µM NAD(H) at 340 nm. Assays were conducted in sodium phosphate buffer (100 mM, pH 7.0) at 30 °C using F96 microwell plates (Nunc, Roskilde, Denmark) and a Multiscan™ FC Photometer (Thermo Fisher Scientific, Waltham, MA, USA). Reactions were initiated by the addition of 10 mM maleimide (ER) or 250 mM sodium formate (FDH) as a substrate. Reaction rates were determined by automated linear regression using MATLAB R2015b (The MathWorks, Natick, MA, USA).
4
Mitochondrial Viability Assay in SH-SY5Y Cells
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Cell viability was evaluated by the reduction of 3-(4,5-di-methyl-thiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an index of mitochondrial functional activity. Briefly, SH-SY5Y cells were seeded into 96 well plates at a density of 20,000 cells/well in complete growth medium for one day and RA differentiated. After differentiation, cells were treated with or without CdCl2 after the pre-treatment with CBD or αToco. After removing the medium with different stimuli, 1 mg/ml MTT was added into each well and incubated for at least 20 min at 37 °C. Following this, the chromogenic solution was removed and replaced with 50 µL of dimethyl sulfoxide (DMSO) to dissolve the formazan crystals, and the absorbance was measured at 595 nm wavelength by a Multiscan FC photometer (ThermoFisher Scientific, Milano, Italy). Three independent experiments were performed, and each experiment was performed in quintuplicate.
5
Cytotoxicity Evaluation of Porous Ceramics
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The cytotoxicity of porous ceramics was determined using a standard method—MTT test (colorimetric test) [42 (link)]. The MTT test is based on the measurement of cell viability through metabolic activity using a colorimetric test. MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow water-soluble tetrazolium salt, which is metabolically reduced by mitochondrial succinate dehydrogenase (SDH) from viable fibroblasts, producing formazan products (blue–violet salt), which cannot cross plasma membranes and accumulates in cells. The number of viable fibroblasts correlates with the color intensity determined photometrically by dissolving formazan in dimethyl sulfoxide (DMSO). To assess cell proliferation on the ceramic surface, fibroblast viability analysis was performed after 24 and 48 h of incubation. The optical density of dissolved formazan was measured using a Multiscan FC photometer (ThermoFisher Scientific, Dreieich, Germany) at a wavelength of 570 nm. As a control, a glass coverslip with high adhesive surface properties, which favor active cell proliferation, was used.
6
Cadmium Toxicity Evaluation via MTT Assay
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Cell viability was evaluated by the reduction of 3-(4,5-di-methylthiozol-2-yl)-2,5diphenyltetrazolium bromide (MTT) as an index of mitochondrial functional activity. Briefly, SH-SY5Y cells were seeded into 96 well plates at a density of 20,000 cells/well in complete growth medium for 1 day. Differentiated and undifferentiated cells were treated with CdCl2 (for 24 h) in the absence or after the pre-treatment with ZnCl2 or Na2SeO3 (for 24 h). After removing the medium with different stimuli, 1 mg/ml MTT was added into each well and incubated for at least 20 min at 37 °C.
Following the removing of the chromogenic solution, the formazan crystals were dissolved in 50 µl of dimethyl sulfoxide (DMSO) and the absorbance was measured at 595 nm by a Multiscan FC photometer (ThermoScientific, Milan, Italy). Three independent experiments were conducted and each experiment was performed in quintuplicate.
Following the removing of the chromogenic solution, the formazan crystals were dissolved in 50 µl of dimethyl sulfoxide (DMSO) and the absorbance was measured at 595 nm by a Multiscan FC photometer (ThermoScientific, Milan, Italy). Three independent experiments were conducted and each experiment was performed in quintuplicate.
7
ELISA-Based Mumps Serology Evaluation
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For serology the ELISA kits RIDASCREEN Mumpsvirus IgM and IgG (R-Biopharm®, Germany) were used according to manufacturer's instructions. Tests contained positive and negative control and calibrators for semi-quantitative evaluations. Final sample photo-absorption was measured on a Multiscan FC photometer (Thermo Scientific®). Measured absorbance values were recalculated according to manufacturer's instructions in units per milliliter (U/ml). Positive results in both tests were set by the manufacturer as a value of more than 24 U/ml and equivocal results in the range from 14.0 to 24.0 U/ml.
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