Rabbit polyclonal anti-DMPO antibody was used to detect DMPO-protein-derived nitrone adducts using a standard ELISA as described previously [31 (link)]. In brief, brain homogenates (nigrostriatal region only) were immobilized by incubating reaction mixtures (2.5 mg/well) in 100 mM bicarbonate buffer, pH 9.6, for 90 min at 35°C. The plate was washed once and blocked (4% fish gelatin in PBS) for 90 min at 35°C. Following another wash, rabbit polyclonal anti-DMPO antibody (1:5,000) was added and incubated for 60 min. After incubation, plate wells were washed four times followed by incubation with anti-rabbit, IgG-alkaline phosphatase-conjugated secondary antibody (1:5,000) for 60 min at 35°C. After another four washes, CDP star (25 mM) was added and the chemiluminescent product was detected using a Tecan Spectra Fluor-Plus multifunction plate reader with Xfluor software (Tecan US, Research Triangle Park, NC, USA).
Xfluor software
Manufactured by Tecan
Sourced in United States
The XFluor® software is a data analysis tool designed for use with Tecan's fluorescence-based assay instrumentation. It provides functionality for acquiring, visualizing, and interpreting fluorescence data generated during experimental workflows.
Automatically generated - may contain errors
Lab products found in correlation
Spectra fluor plus instrument, by Tecan (3 mentions)
Half area 96 well microplates, by Corning (1 mentions)
Adenosine diphosphate (adp), by Promega (1 mentions)
Adenosine triphosphate (atp), by Promega (1 mentions)
Adp glo reagent, by Promega (1 mentions)
Adp glo, by Promega (1 mentions)
Amplex red assay, by Thermo Fisher Scientific (1 mentions)
Spectrafluor plus, by Tecan (1 mentions)
7 protocols using xfluor software
1
DMPO-Protein Adduct Detection via ELISA
2
Colorimetric Nitrite/Nitrate Assay
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The concentration of nitrite/nitrate was determined with the Saville-Griess assay adapted for microtiter plates. A standard curve was prepared with serial dilutions (0–50 µM) of a freshly prepared sodium nitrite (NaNO2) stock solution (100 mM). Cells were homogenized in 1× PBS and, after centrifugation, 200 µl of the supernatant were added to a well of a 96-well plate. Fifty microliters of sulfanilamide solution (4 mg/ml in 1 N HCl) were added to standards and samples. After 2-min incubation, 50 μl of N-(naphtyl)-ethylenediamine dihydrochloride solution (6 mg/ml in H2O) were added, followed by incubation for 5 min at room temperature and measuring absorbance at 540 nm with a Spectra Fluor Plus® instrument and XFluor® software (Tecan, Crailsheim).
3
Nitrite/Nitrate Quantification using Saville-Griess Assay
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The concentration of nitrite/nitrate was determined with the Saville-Griess assay [122] in a microtiter plate. A standard curve was prepared with serial dilutions of freshly prepared 40 μM NaNO2. One hundred micrograms of protein were added to a well and adjusted to 100 µl. 30 µl of 0.5% ammonium sulfamate (ASM; Aldrich) in water was added, followed by 30 µl of 2 N HCl, and incubation for 1–2 min. Subsequently, 40 µl of Griess reagent (sulphanilamide/N-1-napthylethylenediamine dihydrochloride (Promega) was added and incubated for 10 min at room temperature and the absorption at 595 nm was then measured using Spectra Fluor Plus® instrument and XFluor® software (Tecan, Crailsheim).
4
Recombinant human ALK kinase assay
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N-terminal 6His tagged recombinant human ALK expressed in baculovirus Sf21 was purchased from EMD Millipore (Billerica, MA, USA) (purity ≥60% by sodium dodecyl sulfate polyacrylamide gel electrophoresis) and aliquoted to 1 μL fractions when it was used the first time (to avoid multiple freeze/thaws for subsequent experiments). BNP7787 was prepared by a proprietary method (purity >97%, no mesna was detected by mass spectroscopy). Kinase inhibitor, PF02341066 (crizotinib), was purchased from Selleck Chemicals, LLC. (Houston TX, USA). Polyglutamate-tyrosine (PolyGT) substrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Kinase assay buffer was prepared and consisted of 20 mM HEPES, 0.1% Brij 96, 10 mM NaF, 1 mM Na3VO4, and 10 mM MnCl2 adjusted to a final pH of 7.5. Half-area 96-well microplates were purchased directly from Corning Incorporated (Corning, NY, USA). ADP-Glo reagents were purchased from Promega (Madison WI, USA) and consisted of ADP, ATP, ADP-Glo, kinase detection reagent buffer, and kinase detection substrate. All other reagents were purchased from Sigma-Aldrich Co (St Louis, MO, USA). A Tecan Ultra microplate reader with XFluor software (V4.51; Tecan [Morrisville, NC, USA]) and RdrOle software (V4.50; Tecan) were used in this study.
5
IL-8 Protein Secretion Quantification
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To assess the secretion of IL-8 proteins culture medium was collected and subjected to analysis according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA). Absorbance was measured at 450 nm with the use of a Tecan Sunrise spectrophotometer with X-fluor software (Tecan, Männedorf, Switzerland).
6
Hydrogen Peroxide Measurement via Amplex Red
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The Amplex Red assay (Life Technologies) was used to determine levels of H2O2 according to the manufacturer's instructions. Twenty‐four hours after stimulation, the supernatants of 50 000 cells per well were incubated with 50 μL Amplex® Red substrate for 30 minutes in the dark at room temperature, followed by fluorescence measurements at 540/595 nm excitation/emission on the Spectra Fluor Plus® instrument with XFluor® software (Tecan, Crailsheim).
7
Nitrite/Nitrate Quantification Assay
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The concentration of nitrite/nitrate was determined with the Saville‐Griess assay adapted for microtiter plates. A standard curve was prepared with serial dilutions (0‐50 μmol/L) of a freshly prepared sodium nitrite (NaNO2) stock solution (100 mmol/L). Two hundred microlitre of the collected BMDM supernatants was added to a well of a 96‐well plate. Fifty microlitre of sulphanilamide solution (4 mg/mL in 1 N HCl) was added to standards and samples. After short incubation of 1‐2 minutes, 50 μL of N‐(naphthyl)‐ethylenediamine dihydrochloride solution (6 mg/mL in H2O) was added followed by incubation for 5 minutes at room temperature and measuring absorbance at 540 nm with a Spectra Fluor Plus® instrument and XFluor® software (Tecan).
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