CRT (1 µg/mL) diluted in carbonate buffer was added to Polysorp microtiter plates (Thermo–Fisher, Waltman, MA, USA) and incubated overnight (ON) at room temperature (RT). Wells were rinsed with TTN (200 µL/well) and blocked in TTN buffer for 1 h (h) at RT on a shaking table. CRT mAb (1 µg/mL), diluted in TTN buffer, were added to the microtiter wells and incubated for 1 h at RT on a shaking table. Hereafter, the wells were rinsed with TTN (200 µL) for 1 min, and this was repeated 3 times. Next, AP-conjugated goat anti-mouse IgG (1 µg/mL) was added and incubated for 1 h at RT followed by washing as above. Bound Abs were quantified using 1 mg/mL p-NPP in AP substrate buffer, 100 µL per well. The absorbance was measured at 405 nm, with background subtraction at 650 nm on a ThermoMax Microtiter Plate Reader from Molecular Devices (San Jose, CA, USA). All samples were tested in duplicates and corrected for background noise.
Polysorp microtiter plates
Manufactured by Thermo Fisher Scientific
Sourced in United States, Denmark
Polysorp microtiter plates are high-quality polystyrene plates designed for use in various laboratory applications. They feature a uniformly flat surface and a well depth of 11 mm, providing a consistent environment for cell culture, immunoassays, and other experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Softmax pro 3, by Molecular Devices (3 mentions)
Thermo max microtiter plate reader, by Molecular Devices (1 mentions)
Human fibronectin, by Serva Electrophoresis (1 mentions)
Bovine serum albumine (bsa), by Merck Group (1 mentions)
Softmax pro 5, by Molecular Devices (1 mentions)
Spectramax 340pc384, by Molecular Devices (1 mentions)
7 protocols using polysorp microtiter plates
1
Quantitative ELISA for Calreticulin
2
Quantifying Fibronectin-Collagen IV Binding
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Polysorp™ Microtiter plates (Thermo Scientific) were coated with a solution of 25 μg/ml of fibronectin/collagen IV in 100 mM Tris-HCl buffer, pH 8.0, overnight at 4 °C. Unbound protein was removed by washing the plates eight times with PBS containing 0.05% Tween and was subsequently blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature. The blocking buffer was removed, and the wells washed five times prior to the addition of 100 μl protein (10 μg/ml) followed by incubation for 3 h at room temperature. Anti-Agg5A antiserum raised in rabbits (diluted 1:2000) was used to detect the bound protein and anti-rabbit horseradish peroxidase conjugate (1:1000) (Agilent Technologies, Santa Clara, CA) was added following another wash step with PBS + Tween. The peroxidase activity was detected with the addition of TMB plus solution (KPL, Gaithersburg, MD). Optical densities were read at 450 nm with a 96-well plate reader. To analyze the binding data, the background absorbance from wells only containing the protein buffer was subtracted from the absorbance in the test wells.
3
Measuring Ab Isotypes in Prdm1 Mutant Mice
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Ab isotypes were measured by ELISA using serum isolated from Prdm1fl/fl;K14Cre+ and Prdm1fl/+;K14Cre+ mice or from nude hosts grafted with embryonic (E15) thymus isolated from Prdm1fl/fl;K14Cre+ and Prdm1fl/+;K14Cre+ mice. Ninety-six-well Polysorp microtiter plates (Nunc) were coated overnight with a 9:1 ratio mixture of anti-κ: anti-λ (3 μg/ml) (Southern Biotechnology) in 0.1 M Na2HPO4/NaH2PO4 (pH 9). Subsequently, a 1:200,000 dilution of sera was incubated for 2 h at RT. Alkaline phosphatase–labeled goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgE, or IgM secondary Abs were used for detection (Southern Biotechnology). The optical densities were converted to units based on standard curves with Mouse Ig Panel Kit of IgG1, IgG2a, IgG2b, IgG3, IgM, and IgE (Southern Biotechnology) using a four-parameter logistic equation (Softmax Pro 3.1 software; Molecular Devices).
4
Influenza-specific Antibody Detection
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96-well Polysorp microtiter plates (Nunc) were coated overnight with UV-inactivated WSN-GP33/66 in carbonate buffer. AP-conjugated goat anti-mouse IgG and IgA secondary antibodies were used for detection (Southern Biotech). ODs were converted to units based on standard curves with sera from C57BL/6 mice infected with influenza (Softmax Pro 3.1 software; Molecular Devices).
5
Adhesion of Staphylococci to Fibronectin
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Adhesion of tested bacterial strains to human fibronectin was evaluated according to Peacock et al. (2000) (link). All tested strains of staphylococci were preincubated for 24 h and 48 h in the presence of subinhibitory concentrations of chalcones in 24 well plates as previously described in the section of production of glycocalyx. Each isolate had its own positive control (bacteria cultivated solely in medium at the same laboratory conditions).
Polysorp microtiter plates (NUNC, Denmark) were coated for 1 h at 37 °C with 25 μg/mL of human fibronectin (Serva, Germany). After overnight blockage of the remaining sites at 4 °C with 200 μL of 2% bovine serum albumine (Sigma-Aldrich) in PBS, plates were washed three times with PBS and inoculum of 1×108 bacteria in PBS/well was added in triplicates to each well. Plates were incubated 1 h at 37 °C, rinsed three times with PBS, fixed with 2% glutaraldehyde (TCI Europe) in PBS for 1 h and stained with crystal violet for 5 min. After rinsing with water and air drying, the absorbance was measured at OD405. Two triplicates of PBS without bacteria served as a negative control for each plate. After calculation of the results, absorbance values were expressed as the percentage of the absorbance of the positive control. Each assay was repeated three times.
Polysorp microtiter plates (NUNC, Denmark) were coated for 1 h at 37 °C with 25 μg/mL of human fibronectin (Serva, Germany). After overnight blockage of the remaining sites at 4 °C with 200 μL of 2% bovine serum albumine (Sigma-Aldrich) in PBS, plates were washed three times with PBS and inoculum of 1×108 bacteria in PBS/well was added in triplicates to each well. Plates were incubated 1 h at 37 °C, rinsed three times with PBS, fixed with 2% glutaraldehyde (TCI Europe) in PBS for 1 h and stained with crystal violet for 5 min. After rinsing with water and air drying, the absorbance was measured at OD405. Two triplicates of PBS without bacteria served as a negative control for each plate. After calculation of the results, absorbance values were expressed as the percentage of the absorbance of the positive control. Each assay was repeated three times.
6
IgG Quantification of Anti-Na-GST-1 in Mice
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A qualified indirect ELISA described by Jariwala et al [15 (link)] and Bethony et al [17 (link), 18 (link)] was used to measure levels of IgG against Na-GST-1 in murine serum samples. Ninety-six-well Polysorp microtiter plates (NUNC) were coated with 1 μg/mL of recombinant Na-GST-1 onto which experimental mouse sera were also added. Colorimetric reactions were read at a wavelength of 492nm on a SpectraMax 340PC384 (Molecular Devices) using SOFTmax Pro 5.4 for Windows for data capture and analysis.
7
LCMV Antibody Detection by ELISA
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