PON1 arylesterase activity was kinetically measured by following the hydrolysis of phenyl acetate at 270 nm using SpectraMax 190 Absorbance Plate Reader (Molecular Devices LLC, San Jose, USA) as follows: The reagent was prepared using 0.9 mM CaCl2 and 20 mM Tris with vigorous stirring at pH 8.0 w/HCL. A total of 5 µL of serum diluted in 1:5 in assay buffer was pipetted per well in triplicate to a 96-well UV flat bottom plate (Thermo 8404, Thermo Fisher, Waltham, USA). The substrate, phenyl acetate 3.4 µL, was mixed into 25 mL of arylesterase assay buffer and mixed vigorously. Following this, 200 µL of this mixture was added per well using a multichannel pipette and placed in a temperature controlled plate reader SpectraMax 190 Absorbance Plate Reader (Molecular Devices LLC, San Jose, USA) set at 25 °C. The samples were read kinetically at 270 nm for 1.5 min, every 13 s.
Spectramax 190 absorbance plate reader
Manufactured by Molecular Devices
Sourced in United States
The SpectraMax 190 Absorbance Plate Reader is a versatile laboratory instrument designed for measuring the absorbance of samples in microplates. It is capable of performing a wide range of absorbance-based assays, including colorimetric, enzymatic, and cell-based assays, across a wavelength range of 190 to 850 nanometers.
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Lab products found in correlation
Thermo 8404, by Thermo Fisher Scientific (2 mentions)
Tissuelyser adapter set 2 24, by Qiagen (1 mentions)
Human igfbp 3 quantikine elisa kit, by R&D Systems (1 mentions)
Softmax pro software 5.4, by Molecular Devices (1 mentions)
Human igf 1 igf 1 quantikine elisa kit, by R&D Systems (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
96 well plate, by Corning (1 mentions)
Mtt solution, by Merck Group (1 mentions)
Napo4, by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (1 mentions)
Na edta, by Merck Group (1 mentions)
Hexadecyltrimethylammonium bromide htab, by Merck Group (1 mentions)
Sodium chloride (nacl), by Merck Group (1 mentions)
Blocking reagent, by Roche (1 mentions)
Softmax pro software, by Molecular Devices (1 mentions)
96 well microtiter plate, by Corning (1 mentions)
10 protocols using spectramax 190 absorbance plate reader
1
PON1 Arylesterase Activity Assay
2
Measuring Chronic Ethanol Intake in Mice
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In a separate cohort of mice treated identically to the chronic two bottle choice groups, blood ethanol concentrations were determined. Mice were allowed to drink normally except one day before the end of the month-long drinking period or after one week of withdrawal, tail vein blood samples were collected three hours into the dark cycle when mice consume greater amounts of ethanol [31 (link),32 (link)]. The tail bloods were collected into heparinized capillary tubes and sealed with parafilm while all blood samples were collected (<1 h). The capillary tubes were then centrifuged in a hematocrit centrifuge at 10,000 rpm for 5 min and the plasma was used to determine blood ethanol concentration using the Pointe Scientific Alcohol Reagent Kit according to the manufacturer’s protocol and a SpectraMax 190 Absorbance Plate Reader (Molecular Devices).
3
Serum PON1 Lactonase Activity Assay
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PON1 lactonase activity was kinetically measured using 1mM dihydrocoumarin (DHC) as a substrate at and recorded at 270 nm using a SpectraMax 190 Absorbance Plate Reader (Molecular Devices LLC, San Jose, USA) as follows: 10 µL of serum diluted 1:5 in assay buffer (50 mM Tris HCL at pH 8.0 and 1 mM CaCl2) and added per well in triplicate to a 96-well UV flat bottom plate (Thermo 8404, Thermo Fisher, Waltham, USA) The substrate, DHC (8.0 M), was mixed 1:1 in dimethyl sulfoxide (DMSO), vortexed and 6.25 µL of this was added to 25 mL of room temperature lactonase assay buffer and used within 5 min. A total of 200 µL of this mixture was added per well using a multichannel pipette and placed in the temperature controlled plate reader at 37 °C. Samples were read at 270 nm for 2 min 20 s, every 10 s.
Activities were reported as units per liter, where 1 U is defined as 1 mmol of substrate hydrolyzed per minute. Enzyme kinetics was calculated using SOFTmax PRO software version 5.4.6 (Molecular Devices LLC, San Jose, USA).
Activities were reported as units per liter, where 1 U is defined as 1 mmol of substrate hydrolyzed per minute. Enzyme kinetics was calculated using SOFTmax PRO software version 5.4.6 (Molecular Devices LLC, San Jose, USA).
4
Protein Extraction from Agroinoculated Leaves
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Agroinoculeted leaves were homogenized with Tissue Lyser Adapter Set 2 × 24 (QIAGEN, Germany) according to the protocol recommended with the kit. We modified the protocol for the preparation of protein extraction buffer that was previously described [30 (link)]. Briefly, 400 mg of grounded material was extracted with 2 ml of ice-cold 20% TCA/acetone supplemented with 20 mM DTT. The samples were maintained at −20 °C for 30 min and subsequently centrifuged at 16,000 rpm and 0 °C for 20 min. The supernatant was discarded, and the pellet was gently resuspended on 2 ml of ice-cold acetone supplemented with 20 mM DTT. The washing process with acetone was performed twice at −20 °C for 30 min, with centrifugation at 16,000 rpm and 0 °C for 20 min. Finally, the collected pellet that was deprived of chlorophyll was air-dried, and the total protein were extracted with 700 μl of protein extraction buffer (50 mM sodium phosphate (pH 7), 50 mM ascorbic acid, 10 mM di-sodium EDTA, and 1 mM PMSF). The protein concentration was assayed based on the method developed [31 (link)] using a SpectraMax® 190 Absorbance Plate Reader (Molecular Devices, USA).
5
Measurement of IGF-1 and IGFBP-3 in T1DM and Healthy Serum
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IGF-1 and IGFBP-3 levels were measured from T1DM and HC serum samples that were obtained from the MERIT study using the Human IGF-I/IGF-1 Quantikine® ELISA Kit (R & D Systems, Minneapolis, MN, USA) and Human IGFBP-3 Quantikine® ELISA Kit (R & D Systems), respectively, according to the protocol set by the manufacturer. Serum samples underwent a 100-fold dilution in Calibrator Diluent RD5-18. SpectraMax® 190 absorbance plate reader (Molecular Devices, San Jose, CA, USA) and SoftMax® Pro Software 5.4 (Molecular Devices) were used to analyze the data from both the IGF-1 and the IGFBP-3 assays. A standard curve was created by generating a log/log curve fit. Both the IGF-1 and the IGFBP-3 assays were read at 450 nm with correction at 570 nm.
6
Cytotoxicity Assay of SPD Treatment
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Cells were seeded at a density of 1 × 104 cells/well in a 96-well plate (Corning, New York, USA). After overnight incubation, cells were treated with 0, 2, 5, 10, 20, 50, and 100 μM of SPD for 24 h. Subsequently, 100 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reagent was added to the medium in each well and incubated for 3 h at 37 °C. The reduced formazan crystals were solubilized in 50 μL of dimethyl sulfoxide (Sigma-Aldrich) and absorbances were measured at 570 nm in a microplate reader (SpectraMax 190 Absorbance Plate Reader; Molecular Devices, Sunnyvale, CA, USA).
7
MTT Assay for Cell Viability
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Cell viability was assessed by the MTT assay. Briefly, 1.5 × 105 cells were seeded in 6-well plates, incubated for 24 h, and then treated with 2'-FL (10 or 20 g/L) for 24 h. The medium was then replaced by 1 mL of MTT solution (Sigma-Aldrich) for 4 h. The cells were collected, washed, and resuspended in 800 µL of DMSO. The optical density (absorbance) was measured at 570 nm using SpectraMax 190 Absorbance Plate Reader (Molecular Devices, San Jose, CA, USA).
8
Quantifying Neutrophil Levels in Tissue
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The neutrophil dosage in the lesion was performed using the myeloperoxidase assay [78 ]. Tissue samples from Sample 2 were washed with sterile PBS. The supernatant was used for CFU determination, and the washed tissue samples were used for MPO determination. In order to demonstrate that the tissue previously washed with PBS did not affect the MPO measurement, validation assays were performed (data not shown). Biopsies were thawed and buffered in 0.1 M NaCl (Sigma-Aldrich), 0.02 M NaPO 4 (Sigma-Aldrich), 0.015 M NaEDTA (Sigma-Aldrich), and pH 4.7 at –70 °C. Afterwards, buffered tissue samples were triturated in POLYTRON PT 3100 (Avantor) at 13,000 rpm, centrifuged for 15 min at 5000× g, and resuspended in 0.05 M NaPO4 (Sigma-Aldrich) buffer (pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide (HTAB) (Sigma-Aldrich). A total of 25 μL of the supernatant and 25 μL of TMB (3, 3′, 5, 5′-tetramethylbenzidine) (Sigma-Aldrich) were added to each well of a 96-well plate. Subsequently, 100 μL of 0.5 mM H2O2 was added to each well, and the reaction was quenched with 4 M sulfuric acid. MPO activity was measured in a plate reader (SpectraMax® 190 Absorbance Plate Reader, Molecular Devices) at 450 nm. Results are reported as the total number of neutrophils/mg tissue by comparing the absorbance of the tissue supernatant to a standard curve.
9
Monoclonal Antibody-based Gonococcus Detection
10
Ferricyanide Reducing Power Assay
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The reducing power of samples was measured as described by Siddhuraju and Becker [14] with slight modifications. 0.1 mL of sample was dissolved in 0.25 ml phosphate buffer (0.2 M, pH 6.6), mixed with 0.25 mL potassium ferricyanide (1% w/v) and incubated at 50ºC for 20 min. Next, 0.25 mL of trichloroacetic acid (10% w/v) was added and mixed thoroughly followed by centrifugation at 650xg for 10 min. 100µl of distilled water and 25µl of ferric chloride (0.1% w/v) were added to 100µl of supernatant on a 96 well plate and mixed for 10s. The absorbance was read spectrophotometrically at 700 nm after 10 min, using a SpectraMax® 190 Absorbance Plate Reader (Molecular Devices, CA, USA). Measurements were taken in quadruplicate.
The results are expressed in as optical density (OD) at 700 nm. A higher absorbance of the reaction mixture indicated greater reducing power.
The results are expressed in as optical density (OD) at 700 nm. A higher absorbance of the reaction mixture indicated greater reducing power.
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