The changes in proinflammatory cytokines among the four groups were quantified using the Mouse Cytokine Array Panel A (R&D Systems), according to the manufacturer’s instructions. In this experiment, two plantaris samples from a same group were mixed into one sample; thus, there were three samples for each group. Data were normalized to the reference spots of the same membrane. The ratio of control-ex to control-sed and TLR4m-ex to TLR4m-sed were then calculated to compare them.
Mouse cytokine array panel a
Manufactured by R&D Systems
Sourced in United States
The Mouse Cytokine Array Panel A is a multiplex immunoassay that allows for the simultaneous detection and quantification of 40 mouse cytokines, chemokines, and other soluble proteins in a single sample. The panel uses a sandwich immunoassay format and can be used to profile the relative levels of multiple targets in mouse serum, plasma, cell culture supernatants, and other biological samples.
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78 protocols using mouse cytokine array panel a
1
Quantifying Proinflammatory Cytokine Changes
2
Multiplex Cytokine Profiling in Mice
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The Mouse Cytokine Array Panel A from R&D Systems (ARY006, Minneapolis, MN, USA) was used to assess epidermal cytokine protein levels. This array has 40 target cytokines including 17 interleukins and 15 chemokines. Briefly, tissue lysates were homogenized in PBS with protease inhibitors and Triton X-100. Sample protein concentrations were quantified and 300 μg protein or 200 μl of serum was added to the prepared membranes. Next the membranes were incubated with the detection antibody cocktail and washed. Streptavidin-HRP and chemiluminescent detection reagents were added sequentially and the membranes were exposed to X-ray film. Pixel densities were quantified using Image J. Each signal doublet was normalized to the reference signal and then to the signal from TAM67-rTA mice not treated with doxycycline.
3
Quantification of Mouse Cytokine Proteins
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Mouse cytokine protein expressions were quantified using mouse cytokine array (panel A, R&D system, Minneapolis, MN). Briefly, proteins were extracted from Nf1fl/fl;DhhCre mouse neurofibromas and Nf1fl/fl mouse sciatic nerves. Arrays were performed according to the instructions provided by R&D System on 200 μg lysate protein. The intensities of the white dots that were converted from the original black dots were measured using ImageJ software.
4
Cytokine Analysis of Calvarial Bone Defect Healing
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Calvarial defect (4 mm diameter) were treated with a fibrin matrix as described for the calvarial defect model. After 1, 3, 6, 10 and 15 days, the partially remodelled matrix and the bone tissue surrounding the defect (1 mm farther) was collected. As a control a 5 mm diameter calvarial bone tissue was collected (day 0). Fibrinous matrices and tissue samples were incubated in 1 ml of tissue protein extraction reagent (T-PER, Thermo Scientific) containing protease inhibitors (one tablet of protease inhibitor cocktail (Roche) for 10 ml) and homogenized with a tissue homogenizer. Tissue lysates were incubated 1 h at 37 °C and centrifuged at 5,000g for 5 min. Supernatants were stored at −80 °C. Cytokines were detected using an antibody array (Mouse Cytokine Array Panel A, R&D Systems) and by ELISA (Mouse IL-1 beta/IL-1F2 DuoSet and Mouse IL-1ra/IL-1F3 DuoSet, R&D Systems) according to the manufacturer's instructions. For the antibody array, 400 μl of lysate was used. The chemiluminescent signals were detected using ImageQuant LAS 4000 and quantified with ImageQuant TL software (GE Healthcare Life Sciences). A measured volume over 1 × 106 was considered as a positive signal.
5
Cytokine Profiling in Frozen Tissues
6
Hippocampal Cytokine Profiling
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Hippocampal homogenates were analyzed for cytokines presence using a mouse cytokine array panel A (R&D Systems, Minneapolis). A total of 100 μg for each hippocampal lysate were processed following the manufacturer’s instructions.
7
Cytokine profiling post-contusion injury
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Ten days after contusion injury, injured regions were collected and protein was extracted with Illustra triplePrep Kit (GE healthcare). 100 µg of protein lysate were used for the cytokine array (Proteome Profiler antibody assays-Mouse Cytokine Array Panel A, R&D Systems). Images were captured with a Gel Doc XR system (Bio-Rad), and analyzed with ImageJ.
8
Cytokine Secretion Profiling of Msh2-deficient Cells
9
Serum Cytokine Profiling in Mouse Models
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Serum samples from 5XFAD, Tg2576, and their WT control female mice (6 ~ MO) were collected and used for serum cytokine detection. Cytokines were measured with Mouse Cytokine Array Panel A (R&D Systems, cat#ARY006, Minneapolis, MN, USA). Briefly, the mixture of serum and the biotinylated detection antibodies cocktail is incubated with a Mouse Cytokine Array membrane. Any cytokine/detection antibody complex present was bound on the membrane by its cognate immobilized capture antibody. After washing off the unbound protein, streptavidin–horseradish peroxidase and chemiluminescent detection reagents were added to generate light signals. The light produced at each spot is proportional to the amount of cytokine binding.
10
Immunoblotting and Cytokine Profiling
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Immunoblotting was performed using standard protocols. Briefly, the cells were lysed with 1% SDS lysis buffer, and the protein concentration was determined using a BCA assay kit (Pierce). Protein samples were resolved on SDS-PAGE and then transferred to PVDF membranes. The membranes were blocked with 5% BSA, incubated sequentially with primary and secondary antibodies, and then washed. Protein expression was determined by using ECL detection reagent (Pierce).
YAP-induced cytokines in TICs were determined using the Mouse Cytokine Array Panel A from R&D Systems according to the manufacturer's instructions. Briefly, mice were irradiated to ablate the immune system 12 h before HDI. At post-injection day 5, the livers were dissected and homogenized. The homogenates were mixed with a cocktail of biotinylated detection antibodies. The sample/antibody mixture was then incubated with the array membrane. After a wash to remove unbound material, streptavidin-HRP and chemiluminescent detection reagents were added sequentially.
YAP-induced cytokines in TICs were determined using the Mouse Cytokine Array Panel A from R&D Systems according to the manufacturer's instructions. Briefly, mice were irradiated to ablate the immune system 12 h before HDI. At post-injection day 5, the livers were dissected and homogenized. The homogenates were mixed with a cocktail of biotinylated detection antibodies. The sample/antibody mixture was then incubated with the array membrane. After a wash to remove unbound material, streptavidin-HRP and chemiluminescent detection reagents were added sequentially.
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