Cells were washed once with PBS and incubated for 8 h with fresh media. Supernatant was collected, cleared by centrifugation, and used immediately. The amount of supernatant used was normalized to cell number and used with the human cytokine array kit (R&D Systems). IRDye 800CW Streptavidin (Rockland) was used as secondary (1:2,000) and arrays were imaged and quantified on an Odyssey Imager.
Odyssey imager
Manufactured by Rockland Immunochemicals
The Odyssey Imager is a fluorescence-based imaging system designed for the detection and quantification of proteins and nucleic acids in a variety of applications, including Western blotting, protein arrays, and DNA gels. The system utilizes near-infrared fluorescence technology to provide high-sensitivity, high-resolution imaging with a wide linear dynamic range.
Automatically generated - may contain errors
Lab products found in correlation
Human cytokine array kit, by R&D Systems (2 mentions)
Irdye 800cw streptavidin, by Rockland Immunochemicals (2 mentions)
Streptavidin hrp, by Jackson ImmunoResearch (1 mentions)
Tgx 4 16 gel, by Bio-Rad (1 mentions)
Microlon f shape ref 655092, by Greiner (1 mentions)
Donkey antimouse irdye680, by Rockland Immunochemicals (1 mentions)
Odyssey blocking buffer, by Rockland Immunochemicals (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
3 protocols using odyssey imager
1
Cytokine Profiling using Array
2
Cytokine Profiling using Array
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Cells were washed once with PBS and incubated for 8 h with fresh media. Supernatant was collected, cleared by centrifugation, and used immediately. The amount of supernatant used was normalized to cell number and used with the human cytokine array kit (R&D Systems). IRDye 800CW Streptavidin (Rockland) was used as secondary (1:2,000) and arrays were imaged and quantified on an Odyssey Imager.
3
Quantification of Amyloid-Beta Levels
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Immunoassay. Microplates Microlon/F-shape REF 655092 (Greiner) were coated with 1 µg/mL of human 3D6 (29) overnight at room temperature in coating buffer (sodium carbonate pH=9.6 0.05 M NaCO 3 in MQ water). After washing plates (washing buffer 0.05% Tween-20 in PBS were blocked with 4% not fat dry milk and incubated with brain homogenates or with Aβ to generate the standard curve. Next plates were washed, incubated with 50 ng/mL of biotinylated human 20C2 (29) and washed again. Finally, plates were incubated with streptavidin-HRP (Jackson ImmunoResearch Laboratories) diluted 1:8,000 and developed using 3,3',5,5'-Tetramethylbenzidine (TMB). The absorption was measured at 450 nm within 30 minutes of stopping the reaction with 2 M H 2 SO 4 using the Perkin Elmer 2030 manager system. Western blot. TBS and TBS-T samples corresponding to 40 µg of total protein we loaded onto a precast TGX 4-16% gel (Bio-Rad). Then samples were transferred to PDVF membranes, blocked with Odyssey blocking buffer (LI-COR Bioscience) and probed with 1 µg / mL mAb anti-Aβ (6E10, Covance). After incubation with donkey anti-mouse IRdye680 (Rockland Immunochemicals) diluted 1:1,000 in Odyssey blocking buffer, the membrane was scanned and analyzed with the Odyssey imager. The intensities of the APP bands detected at 100 kDa, were measured with the Odyssey imager.
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