Mouse cytokine arrays were performed on whole brain homogenates of SAH and sham operated mice three days after hemorrhage. The brains were dissected, homogenized, delipidated and stored in PBS. Samples were centrifuged and the supernatant was stored with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and frozen at −80° until time for evaluation. Samples were tested with a 40-analyte mouse cytokine/chemokine array (Proteome Profiler Array mouse cytokine array panel a [R&D Systems, Minneapolis, MN]) using the manufacturer’s instructions with the exception that Li-Cor compatible secondary antibodies (Licor Biosystems, Lincoln, NE) were used to resolve the membranes with a Li-Cor Odyssey Clx infrared imaging system (Licor Biosystems, Lincoln, NE). Intensity values were corrected for local membrane effects using a circular perimeter around the sample dot. Relative intensity was defined as corrected sample intensity as a ratio of the negative control dot. Data was tabulated and evaluated using Graphpad Prism 7 software (Graphpad, La Jolla,CA, US).
Proteome profiler array mouse cytokine array panel a
Manufactured by R&D Systems
Sourced in United States
The Proteome Profiler™ Array Mouse Cytokine Array Panel A is a laboratory equipment product designed for the analysis of mouse cytokines. It provides a platform to simultaneously detect and quantify the relative levels of multiple cytokines in a single experiment.
Automatically generated - may contain errors
Lab products found in correlation
Imagequant las 4000, by GE Healthcare (3 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Prism 7, by GraphPad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
Supersignal west femto chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Mmcp 1, by R&D Systems (1 mentions)
Mtnf α, by BD (1 mentions)
Image quant tl software version 1.51h, by GE Healthcare (1 mentions)
Supersignal west femto chemiluminescent kit, by Thermo Fisher Scientific (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
11 protocols using proteome profiler array mouse cytokine array panel a
1
Mouse Brain Cytokine Profiling After SAH
2
Serum Cytokine Analysis in Mice
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Blood samples were collected by cardiac puncture immediately before the perfusion and sera were obtained by centrifugation at 2000 × g, at 4°C for 10 min The serum cytokine analysis was performed with pooled serum samples in equal proportions for 14 mice per group, obtaining four replicates for each condition in two independent experiments according to the manufacturer’s instructions. (Proteome Profiler™ Array Mouse Cytokine Array Panel A; R&D Systems; ARY006). Samples were measured on the ChemiDocTM MP Blot reader system (BioRad; Hercules, Calif.). Image Lab Software (BioRad; Hercules, Calif.) was used for data analysis.
3
Cytokine Profiling in Cell Cultures
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300,000 cells were seeded in a six-well plate. 500 μl of media was collected after 72 h. Samples were spun for 10 min at 300 × g to remove any residual cells. The cytokine array was performed using the Proteome Profiler Array Mouse Cytokine Array Panel A (R&D Systems, Minneapolis, MN, United States), following the manufacturer’s protocol. Quantification was done in ImageJ using the Dot Blot Analyzer for ImageJ by Gilles Carpentier, Universite de Paris, France (Carpentier, 2008 ).
4
Cytokine Profiling in Macrophage Supernatants
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Culture supernatants of macrophages mentioned previously were measured for the presence of cytokines using Proteome Profiler™ Array Mouse Cytokine Array Panel A (ARY006; R&D Systems, Inc., Minneapolis, MN, USA). Protein array was performed following protocol supplied by the manufacturers.
5
Cytokine Profiling of DPSC Secretome
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The FBS-free DPSC culture medium was collected after 2 days of incubation for array analysis. The relative expression of cytokines and chemokines in the culture medium was quantified using the Proteome Profiler™ Array (Mouse Cytokine Array, Panel A; R&D Systems Inc.), as previously described [28 (link)]. To achieve maximum assay sensitivity, the blots were incubated overnight with plasma. Enhanced chemiluminescence incubation was performed for 5 min using the Super Signal West Femto Chemiluminescence Kit (Thermo Scientific Pierce), and the samples were imaged and analyzed using the Image Quant LAS 4000 coupled with Image Quant TL software (GE Healthcare Japan, Tokyo, Japan) and Image J software (NIH, Bethesda, MD).
6
Plasma Cytokine Analysis in Mice
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Blood samples were collected by submandibular vein puncture and plasma was obtained by centrifugation at 2000×g, RT for 10 min. Plasma cytokine analysis was performed with pooled plasma samples at equal ratios for five mice per group according to the manufacturer’s instructions (Proteome Profiler™ Array Mouse Cytokine Array Panel A; R&D Systems; ARY006). Samples were measured on the ChemiDocTM MP Blot reader system (BioRad; Hercules, Calif.). ImageJ 1.48 and the Gilles Carpentier’s Protein Array Analyzer for ImageJ toolset were used for data analysis.
7
Cytokine Profiling in Infected Cells
8
Cytokine Profiling of Murine Lung Homogenates
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Post-lavage lungs, prepared from three mice (10-weeks-old), were homogenized in PBS containing 1% triton X-100 and protease inhibitors (5 mg/ml aprotinin, 10 mM leupeptin, 1 mM pepstatin, and 1 mM phenylmethylsulfonyl fluoride). The homogenates were centrifuged for 20 min at 18,340×g at 4 °C. The resulting supernatants (200 μg of proteins) were used with a Proteome Profiler Array (Mouse cytokine array panel A; R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Chemiluminescence signals on the antibody array were detected using an ImageQuant LAS 4000 image analyzer (GE Healthcare, Little Chalfont, UK).
9
Murine Bone Marrow Cytokine Profiling
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For collecting BM fluids, the 2 femurs and 2 tibiae of each mouse were flushed with 200 μL of Hanks balanced salt solution/2% fetal bovine serum using a 0.3 mL insulin syringe with a 28-gauge needle and centrifuged at 500 × g for 5 minutes to remove the BM cells. The supernatants were further clarified by centrifugation at 12 000 × g for 10 minutes, and samples were subsequently stored at −80°C until use.29 (link) Cytokine levels were determined using the Proteome profiler array, mouse cytokine array panel A (R&D #ARY006) according to the manufacturer’s instructions. Each loaded sample was combined from 2 mice.
10
Cytokine and Chemokine Quantification in Muscle Tissue
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Muscle tissue samples were disrupted in a homogenizer (Psychotron NS-310EII; Microtec Co, Ltd, Chiba, Japan), and the protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Life Science, Hercules, CA). The relative expression of cytokines and chemokines in muscle lysates was quantified using the Proteome Profiler Array (Mouse Cytokine Array, Panel A; R&D Systems Inc., Minneapolis, MN). The array was performed according to the manufacturer's specifications with 100 mg of total protein. To achieve maximum assay intensity, the blots were incubated overnight. The chemoluminescence signal was generated by incubation with the Super Signal West Femto Chemiluminescent Kit (Thermo Scientific Pierce, Rockford, IL) for 5 minutes. Images were captured using Image Quant LAS 4000 coupled with Image Quant TL software version 1.51h (GE Healthcare, Tokyo, Japan).
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