Relative levels of several cytokines were assessed with the “Mouse Cytokine Array Panel A Array kit” (R&D Systems). Mouse plasma was tested as recommended by the manufacturer. Signal intensity was quantified by ImageJ software and data expressed as a percentage of intensity normalized to positive controls.
Mouse cytokine array panel a array kit
Manufactured by R&D Systems
Sourced in United States
The Mouse Cytokine Array Panel A Array Kit is a multiplex sandwich immunoassay used to detect the relative levels of 40 different mouse cytokines, chemokines, and other soluble proteins in a single experiment. The array utilizes capture and detection antibodies spotted in an array format on a membrane. Samples are incubated with the array, and bound proteins are detected using a chemiluminescent readout. This kit provides a convenient and sensitive method to profile the expression of multiple proteins simultaneously in mouse biological samples.
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8 protocols using mouse cytokine array panel a array kit
1
Cytokine Profiling in Mouse Plasma
2
UV-Induced Cytokine Profiling in Mice
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Pro-inflammatory cytokine induction as a result of UVR was monitored using the Mouse Cytokine Array Panel A Array Kit (ARY006, R&D Systems) as recommended by the manufacturer. Briefly, SKH-1 mice were unirradiated (control) or were irradiated with 500 J/m2 of UV either at ZT 21 (4 AM) or ZT 9 (4 PM), and whole skin was collected 12 hrs after UVR and snap frozen. Protein lysates were prepared with tissue homogenization using a mortar and pestle in liquid nitrogen followed by suspending the powdered sample in 1× PBS with protease inhibitors and 1% Triton X-100. Clear protein lysates (250 μg) were then probed using the mouse cytokine antibody to monitor cytokine levels.
3
UV-Induced Cytokine Profiling in Mice
4
Detecting Soluble Proteins in MSCs
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To detect soluble protein, MSCs Pml+/+ or Pml−/− were plated at the same numbers, and the supernatant was collected after a few days of culture in hypoxic conditions. Collected supernatants were analyzed within Mouse Cytokine array panel A array kit and Angiogenesis array kit (R&D Systems), following manufacturer instructions.
5
Mouse Cytokine Array Analysis
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Mouse Cytokine Array Panel A Array kit (R&D Systems) was used according to the manufacturer’s instructions. In brief, 80–100 µl of sera previously mixed with antibody cocktail was added to the precoated membranes and incubated overnight. The membranes were incubated with streptavidin. The results were visualized by ImageQuant imaging system and analyzed with the ImageJ software (National Institutes of Health) with dot blot analysis plug-in.
6
Comprehensive Blood Analysis Protocol
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Blood samples were collected using a disposable anticoagulant tube containing EDTA and analyzed using an Element Ht5 Auto Hematology analyzer (Heska, Canada) for automatic Complete Blood Count within 6 h of collection. Blood samples were collected using disposable 1.5 ml centrifuge tubes for obtaining serum. Relative levels of cytokines and chemokines were determined by using the Mouse cytokine array panel A array kit (ARY006, R&D Systems, Minneapolis, USA). Selected cytokines, including TNF, IL-1β and IL-1RA were measured by ELISA kits (CSB-E04741, CSB-E08054 & CSB-E10395, CUSABIO, Wuhan, China).
7
Quantitative Cytokine Profiling in Tissue Lysates
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Lymph node cells were lysed using RIPA lysis buffer and proteinase inhibitors (Roche) to extract proteins. Protein concentration in the tissue lysates was determined by bicinchoninic acid Protein Assay Reagent (Pierce). Proteins from tissue lysates (70 μg) were analysed using a commercial kit (Mouse cytokine array panel A Array kit, R&D Systems) containing nitrocellulose membranes coated with 40 different anti-cytokine or anti-chemokine antibodies printed in duplicate, following the manufacturer's instructions. Chemiluminescenc was measured by ImageQuantum LAS4000 (GE Healthcare Life Science) and signals quantified using AIDA Evaluation Software (BioImaging).
8
Cytokine Profiling of Serum-Deprived Cells
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Total 0.4 × 106 cells were seeded in 6-well plate, after the cell were attached, cells were serum deprived for 12 h and then treated with different stimuli for 48 h. Cell supernatant were collected and centrifuged at 2000 g for 10 min. And then supernatant were analyzed using a Mouse Cytokine Array Panel A Array Kit (R&D Systems) according to the manufacturer’s instructions. The results were scanned and analyzed using ImageJ software. CXCL10 levels of the cell culture supernatant were measured by ELISA kit according to the manufacturer’s protocol.
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