Serum samples were individually obtained by centrifugation of the whole blood at 8000 × g for 15 min twice. Three different p14 and 20wo serum batches (each a pool from 6 animals) were analyzed using the RayBio Mouse Cytokine Antibody Array Kit (Raybiotech, Inc. Georgia, USA) and Proteome ProfilerTM Array/Mouse XL Cytokine Array Kit (R&D Systems, Minneapolis, USA) following manufacturer’s instructions. Membranes were scanned using a LAS4000 Lumi-Imager (Fuji Photo Film, Valhalla, NY) and analyzed using ImageJ/Fiji software. The results were then normalized using internal controls, and the relative protein abundance in p14 compared to adult mouse serum was represented.
Proteome profiler array mouse xl cytokine array kit
Manufactured by R&D Systems
Sourced in United States
The Proteome Profiler Array Mouse XL Cytokine Array Kit is a multiplex array designed to simultaneously detect the relative levels of 111 mouse cytokines, chemokines, and other soluble proteins in cell culture supernates, tissue lysates, or other biological fluids. The kit includes a nitrocellulose membrane pre-spotted with capture antibodies, as well as all necessary detection reagents.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Las4000 lumi imager, by Fujifilm (1 mentions)
Raybio mouse cytokine antibody array kit, by RayBiotech (1 mentions)
Super rx n, by Fujifilm (1 mentions)
Anti β actin a1978, by Merck Group (1 mentions)
Anti il 17a, by R&D Systems (1 mentions)
Anti il 23, by R&D Systems (1 mentions)
Anti il 23r, by R&D Systems (1 mentions)
Anti mcpip 1, by R&D Systems (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Image studio software, by LI COR (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Gentlemacs dissociator, by Miltenyi Biotec (1 mentions)
Enhanced chemiluminescence method, by GE Healthcare (1 mentions)
Anti il 17a, by Proteintech (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Legend max mouse il 34 elisa kit, by BioLegend (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
7 protocols using proteome profiler array mouse xl cytokine array kit
1
Mouse Serum Cytokine Profiling
2
Cytokine Profiling in EAE Mouse Model
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The Proteome Profiler TM Array -Mouse XL Cytokine Array Kit (R&D Systems, ARY028) was used to determine the relative expression level of cytokines and chemokines in the spinal cord of EAE mice. Experiments were conducted according to the manufacturer's instructions. Cells prepared from spinal cord tissues were lysed in RIPA buffer [50mM Tris-HCl (pH8.0), 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.3% SDS, 1mM PMSF, and 1X protease inhibitor cocktail]. Protein concentration was detected by PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific). 200µg spinal cord protein was incubated with array membrane for overnight at 4°C on a rocking platform shaker. After incubation, the membrane was washed and incubated with the Detection Antibody Cocktail for 1 hour at RT. The membrane was then washed and incubated with 1X Streptavidin-HRP for 30 minutes at RT followed by washing and incubation with Chemi Reagent Mix. Signal dots appeared on the membrane were then detected by X-ray film (FUJIFILM Super RX-N). The fold change of signal dots representing cytokine and chemokine expression was measured and determined by using protein array analyzer running under Image J (National Institutes of Health).
3
Molecular Profiling of Corneal Inflammation
4
Cytokine Profiling in Murine Lung Injury
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Lungs of at least three mice per group (Control, Elastase & Elastase + MSC) were dissociated using a gentleMACS Dissociator (Miltenyi Biotec), protein was measured with the DC™ Protein Assay (Bio-Rad, Hercules, CA, USA), and equal protein concentrations per group were analyzed following the Proteome Profiler™ Array (Mouse XL Cytokine Array Kit, R&D Systems ARY028) recommended protocol (http://bit.ly/2lAbFda ) assessed on 1 May 2022. The membrane spots were quantified using the Image Studio software (Li-Cor Biosciences).
5
Protein Extraction and Analysis from Lung Tissues
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Proteins from lung tissues were extracted using a Nuclear and Cytoplasmic Protein Extraction kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) and protein concentrations were evaluated using a BCA protein assay. For Western blot analysis, A total of 24 μg aliquots of protein samples mixed with a loading buffer were separated by 10% SDS−PAGE and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% milk and subsequently incubated with primary and secondary Abs. The protein bands were illuminated using the enhanced chemiluminescence method (GE Healthcare Life Sciences, Little Chalfont, Buckinghamshire, UK), and quantified by densitometry analyses using ImageJ software version 1.8.0. The primary Abs used included anti-IL-23 (Boster Biological Technology, Wuhan, Hubei, China), anti-IL-23R (Proteintech Group, IL, USA), anti-IL-17A (Proteintech Group, IL, USA), anti-IL-17RA (Servicebio Biological Technology, Wuhan, Hubei, China), anti-RBP4 (R&D Systems, Minneapolis, MN, USA), and anti-β-actin (BioTNT, Shanghai, China). ELISA and Cytokine array (Proteome Profiler Array Mouse XL Cytokine Array Kit; R&D Systems, Minneapolis, MN, USA) were performed following manufacturer’s protocols.
6
Profiling Cytokine Expression in Mouse Calvaria
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After 1 week, the samples (n = 5/per group) harvested from mouse calvaria were homogenized in PBS with protease inhibitor (10 μg/mL of pepstatin, aprotinin, leupeptin), and Triton X-100 was added to a final concentration of 1% and frozen at ≤ −70 °C, thawed, and centrifuge at 10,000 g for 5 min and the supernatant was collected. The supernatants were assayed using the Proteome Profiler Array Mouse XL Cytokine Array Kit (ARY028, R&D Systems) according to the manufacturer's protocol. The array kit contains nitrocellulose membranes spotted with capture antibodies. Each membrane was incubated with supernatant at 4 °C overnight. After extensive washing to remove non-bound proteins, the membranes were incubated with biotinylated detection antibodies. Next, streptavidin–horseradish peroxidase was added to visualize the antibody reactions, which were detected using an image analyzer system (ImageQuant LAS 4000 mini, GE Healthcare).
7
Quantifying CSF1 and IL-34 in Murine Samples
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ELISA assays of CSF1 were performed using the Invitrogen Mouse M-CSF1 (CSF1) ELISA kit (ThermoFisher Scientific) following the manufacturer’s instructions using either cell culture supernatants or mouse sera. IL-34 levels in mouse sera were assessed using the LegendMax Mouse IL-34 ELISA kit purchased from Biolegend.
The Proteome Profiler array Mouse XL Cytokine array kit (R&D Systems) was performed using cell culture supernatants. Briefly, 2 × 105 cells per well were seeded on a 6-well plate. Following a 24-h incubation, the cells were washed with PBS, and media was replaced with RPMI serum-free media. Twenty-four h later, cell supernatant was collected and centrifuged at 500 g for 5 min to remove cell debris. The assay was run using the manufacturer’s recommended protocol and imaged using the LI-COR Odyssey Infrared Imaging System. Image analysis was performed using the ImageJ software.
The Proteome Profiler array Mouse XL Cytokine array kit (R&D Systems) was performed using cell culture supernatants. Briefly, 2 × 105 cells per well were seeded on a 6-well plate. Following a 24-h incubation, the cells were washed with PBS, and media was replaced with RPMI serum-free media. Twenty-four h later, cell supernatant was collected and centrifuged at 500 g for 5 min to remove cell debris. The assay was run using the manufacturer’s recommended protocol and imaged using the LI-COR Odyssey Infrared Imaging System. Image analysis was performed using the ImageJ software.
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