Luminex assay was performed according to the described protocol 21 . At different time points after injury, mice received an intraperitoneal injection of sodium pentobarbital (Dolethal). Blood was removed by perfusion with 60 mL of 0.9% NaCl in distilled water. 0.6 cm of the spinal cord, centered into the injury site, were taken from each mouse. Samples were frozen in liquid nitrogen and homogenized in extraction buffer using a TissueRuptor (Qiagen, #9002755) and an Ultrasonic Homogenizer (Biologics Inc., #3000). Extraction buffer was prepared following the previous publication 21 . Protein was quantified and diluted to 2 mg/µL. Beads and samples were added to the Luminex plate following specific commercial protocols (Invitrogen). After washing, 50 µL of samples were added and incubated overnight at 4ºC. Finally, secondary antibodies were added and the levels of IL-13 and IL-4 were calculated using a MAGPIX Luminex reader (ThermoFisher Scientific). Final values were normalized to the protein concentration of the samples. 4 mice per each time-point were used.
Magpix luminex reader
Manufactured by Thermo Fisher Scientific
The MAGPIX luminex reader is a multiplex assay system that uses magnetic microspheres to simultaneously measure multiple analytes in a single sample. The system utilizes flow cytometry and xMAP technology to detect and quantify target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Antibody isotyping 7 plex mouse procartaplex panel, by Thermo Fisher Scientific (2 mentions)
Single elisa kits, by Thermo Fisher Scientific (2 mentions)
Phosphatase substrate p nitrophanylphosphate disodium, by Merck Group (2 mentions)
Alkaline phosphatase conjugated anti mouse igg, by Merck Group (2 mentions)
Tissueruptor, by Qiagen (1 mentions)
3 protocols using magpix luminex reader
1
Cytokine Profiling in Spinal Cord Injury
2
Antibody Isotype and MDA-Specific IgG Assay
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To measure Ig isotypes and subclasses in plasma, the Antibody Isotyping 7-Plex Mouse ProcartaPlex™ Panel was performed with a MAGPIX luminex reader (ThermoFisher). For total IgG and IgM quantification, single ELISA kits were used (ThermoFisher). Anti-MDA specific IgGs were measured by coating MaxiSorp plates (NuncTM, city, Roskilde, Denmark) with purified, mouse-derived delipidated apolipoprotein MDA for 1 h at 37° C. After being washed, all wells were blocked for 1 h with 2% BSA in a PBS at 37° C. Mouse samples were also added to a non-coated well in order to assess individual non-specific binding. After washing, 50 μl/well of the alkaline phosphatase-conjugated anti-mouse IgG was added (Sigma-Aldrich), it was diluted at 1:1000 in a PBS/BSA 2% solution, and this was added and incubated for 1 h at 37° C. After washing, phosphatase substrate p-nitrophanylphosphate disodium (Sigma-Aldrich) dissolved in a diethanolamine buffer (pH 9.8) was added and incubated for 30 min at 37° C. Optical density (OD) was determined at 405 nm (Filtermax 3, Molecular DevicesTM, San Jose, CA) and each sample was tested in duplicate. Corresponding non-specific binding was subtracted from mean OD for each sample.
3
Antibody Isotype and MDA-Specific IgG Assay
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