Rat cytokine array panel a

Manufactured by R&D Systems

Sourced in United States, United Kingdom

The Rat Cytokine Array Panel A is a multiplex assay that allows for the simultaneous detection and quantification of 29 different cytokines and chemokines in rat biological samples. The kit uses a sandwich immunoassay-based approach to measure the relative levels of each target analyte.

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Rat cytokines array panel A Cytokine Array Panel A Cytokine arrays Panel A

11 protocols using rat cytokine array panel a

Cytokine Profiling in Radiation-Exposed Rats

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An antibody-based cytokine array (Rat Cytokine Array Panel A, R&D Systems, Minneapolis, MN) was used to assess levels of 29 different cytokines in the frontal cortices of 25 cGy-exposed F344 and LEW rats and sham-irradiated controls of both strains. The 25 cGy rats were assessed with the array because it was at this radiation dose where the behavioral deficits were most severe in the F344 rats. The array procedure was carried out according to the manufacturer’s instructions.

Davis C.M., DeCicco-Skinner K.L, & Hienz R.D. (2015). Deficits in Sustained Attention and Changes in Dopaminergic Protein Levels following Exposure to Proton Radiation Are Related to Basal Dopaminergic Function. PLoS ONE, 10(12), e0144556.

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Cytokine Profile Analysis of Spinal Cord Injury

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Cytokines profile of CM derived from control, SCI + V, SCI + CM after 15 days and 30 days for the segment R1 was performed by using a Rat Cytokine Array Panel A according to the manufacturer’s instructions (R&D Systems, Inc., Abingdon, UK). Conditioned media from rostral segments from spinal cord tissue (all experimental groups and control) were obtained according to our previous study and afterwards processed for Cytokine profile detection [25 (link)]. We have used a similar procedure as in our previous study [26 (link)]. Briefly, the array membranes were first incubated in the blocking buffer for 1 h. Afterwards 200 µL of CM were mixed with the detection antibody mixture and incubated for 1 h at room temperature. After removing the blocking buffer, the sample/antibody mixture was added to array membranes and incubated overnight at 4 °C. Next day, the membranes were washed with the buffer and then incubated with Streptavidin-HRP solution for 30 min at room temperature. The membranes were finally washed with buffer 4 times and the bound antibodies were detected by chemoluminescence using a chemireagent mix. The membranes were quantified by densitometry using ImageJ software. Background staining and spot size were analyzed as recommended by the manufacturer. Normalization was done with control expression.

Cizkova D., Cubinkova V., Smolek T., Murgoci A.N., Danko J., Vdoviakova K., Humenik F., Cizek M., Quanico J., Fournier I, & Salzet M. (2018). Localized Intrathecal Delivery of Mesenchymal Stromal Cells Conditioned Medium Improves Functional Recovery in a Rat Model of Spinal Cord Injury. International Journal of Molecular Sciences, 19(3), 870.

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Rat Cytokine Array Assay Protocol

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Rat cytokine array was performed by using commercially available kit (Rat cytokine array panel A, R&D systems). The MLN was collected and homogenized in PBS supplemented with protease inhibitor (1mM PMSF). After homogenization, Triton X-100 was added to a final concentration of 1%. Then the samples were store at −80°C. After thaw and centrifuge at 10000g for 5 min, the supernatant was collected for cytokines detection.

Zhu D., Liu S., Huang K., Wang Z., Hu S., Li J., Li Z, & Cheng K. (2022). Intrapericardial Exosome Therapy Dampens Cardiac Injury Via Activating Foxo3. Circulation research, 131(10), e135-e150.

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Spinal Cord Cytokine Profiling

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Spinal cords were homogenized in ice-cold lysis buffer (25 mM Tris HCl pH 7.4, 15 mM NaCl, 10 mM NaF, 10 mM Na Pyrophosphate, 2 mM EDTA, 0.2 mM Na₄OV₃, 1 mM PMSF, Protease Inhibitor Cocktail (1 : 200; Sigma Aldrich, UK)) using the FastPrep® bead beater system (MP Biomedicals, USA). The protein concentration of each sample was determined using a BCA assay (Pierce Biotechnology, UK) and 350 μg of lysate was loaded per membrane (Rat Cytokine Array Panel A, R&D Systems, UK), as per manufacturer's instructions. Cytokine levels were visualised and quantified using IRDye® 800CW Streptavidin secondary antibody (1/2000 in assay buffer 6; Li-Cor, UK) and captured on an Odyssey® Infrared Imaging System. Results were analysed using Odyssey 2.1 Software (Li-Cor, UK) and all absorbance values were corrected for background.

Newton V.L., Guck J.D., Cotter M.A., Cameron N.E, & Gardiner N.J. (2017). Neutrophils Infiltrate the Spinal Cord Parenchyma of Rats with Experimental Diabetic Neuropathy. Journal of Diabetes Research, 2017, 4729284.

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Cytokine levels in α-Syn overexpression

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Before α-Syn overexpression and Gm-csf mRNA treatment, rats were bled via maxillary vein, and peripheral blood was collected as a baseline. After collection, blood was centrifuged at 1000 x g for 10 minutes, and plasma was collected and stored at −80°C. After 28 days, at the time of sacrifice, blood and plasma were collected again. After collection, levels of cytokines within plasma before and after treatment were assessed using Rat Cytokine Array Panel A (R&D Systems) protocol.

Olson K.E., Namminga K.L., Lu Y., Thurston M.J., Schwab A.D., de Picciotto S., Tse S.W., Walker W., Iacovelli J., Small C., Wipke B.T., Mosley R.L., Huang E, & Gendelman H.E. (2021). Granulocyte-Macrophage Colony-Stimulating Factor mRNA and Neuroprotective Immunity in Parkinson’s Disease. Biomaterials, 272, 120786.

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Prostate Cytokine Profiling in Rats

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One rat prostate sample was randomly selected from each of the NC group, EAP group, EAPK group, and EAPKH group, respectively. An equal amount of frozen prostate tissue was powdered under liquid nitrogen and homogenized in PBS with protease inhibitors. The protein concentration of the tissue homogenate was detected by the BCA protein quantification assay kit. Prostate cytokine protein levels were analyzed using Rat Cytokine Array Panel A (catalog no. ARY008, R&D Systems, USA) according to the manufacturer's instructions. Pixel densities of 13 cytokines with visible differences on the 4 developed X-ray films were analyzed using ImageJ software.

Zhang M., Lin D., Luo C., Wei P., Cui K, & Chen Z. (2022). Tissue Kallikrein Protects Rat Prostate against the Inflammatory Damage in a Chronic Autoimmune Prostatitis Model via Restoring Endothelial Function in a Bradykinin Receptor B2-Dependent Way. Oxidative Medicine and Cellular Longevity, 2022, 1247806.

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Rat Cytokine Array Analysis

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The Rat Cytokine Array Panel A (Cat # ARY008) from R&D system (Minneapolis, MN) was used to probe cytokines in PFP from MSZR and LZR by following the procedures recommended by the manufacturer. Bound antibodies were detected by chemiluminescence using the Immobilon™ Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA). This was performed once with a plasma pool from 5 to 6 animals to reduce inter-animal variability in each group.

Lagrange J., Didelot M., Mohamadi A., Walton L.A., Bloemen S., de Laat B., Louis H., Thornton S.N., Derby B., Sherratt M.J., Fève B., Challande P., Akhtar R., Cruickshank J.K., Lacolley P, & Regnault V. (2017). Implication of Free Fatty Acids in Thrombin Generation and Fibrinolysis in Vascular Inflammation in Zucker Rats and Evolution with Aging. Frontiers in Physiology, 8, 949.

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Quantification of Brain Inflammation

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After 3 days of DBI, we fully perfused the brain tissues with ice‐cold PBS and homogenized the tissue with PBS containing a protease inhibitor. We then centrifuged the samples at 4°C and 10,000 × g for 5 min. We used the Solaribo (BCA) method to determine the total protein concentration in the samples. Inflammatory cytokines, including CXCL‐1, CXCL‐7, CXCL‐9, CXCL‐5, CX3CL1, IL‐1a, IL‐1b, IL‐1ra, IL‐3, IL‐6, IL‐10, TNF‐α, and VEGF, were assessed using Rat Cytokine Array Panel A (ARY008, R&D Systems, Minneapolis, MN, USA) according to the protocol. The protein (200 μg) was mixed with the biotinylated detection antibody and incubated with the membrane at 4°C overnight. The next day, the membrane was washed thoroughly with 1× washing buffer solution and incubated with streptavidin HRP on a shaker at room temperature for 30 min. After further washes, the membrane is exposed to the ECL system (Millipore, Billerica, MA). We repeated these experiments three times. Finally, we measured the expression of inflammatory cytokines and chemokines using Quantity One software (version 4.6.2; Bio‐Rad) and averaged the pixel density. We corrected the spot density of a single background to reduce variation between the arrays.

Feng D., Liu T., Zhang X., Xiang T., Su W., Quan W, & Jiang R. (2024). Fingolimod improves diffuse brain injury by promoting AQP4 polarization and functional recovery of the glymphatic system. CNS Neuroscience & Therapeutics, 30(3), e14669.

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Cytokine Profiling of Stem Cell EVs

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Cytokine arrays were performed using the Rat Cytokine Array Panel A (R&D Systems, ARY008). Briefly, SCs were cultured in 6-well plates and upon reaching 85% confluence, the growth medium was replaced with DMEM containing 1% exosome-free FBS and 100 U/ml penicillin and 100 μg/ml streptomycin (1%). TNFα (0.5 nM) alone or TNFα (0.5 nM) plus SC-EVs (10 μg) was added to the SC cultures. After 3 h, stimulated cells were lysed using the recommended lysis buffer modified by us (1% IGEPAL, 20 mM Tris–HCl [pH 8.0], 137 mM NaCl, 10% glycerol, 2 mM EDTA, and Protease inhibitor) for 30 min at 4°C followed by centrifugation at 14,000 g for 5 min. Cell extracts were incubated with nitrocellulose membranes, pre-loaded with antibodies, as recommended by the manufacturer. Membranes were developed using Thermo Scientific^™ SuperSignal^™ West Femto Maximum Sensitivity Substrate kit (Thermo Scientific, PI34095) on autoradiography films. Densitometry of each dot was performed using ImageJ.

Improving the debate on cannabis. (2000). BMJ : British Medical Journal, 321(7261), 623.

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Cytokine profiling of implanted scaffolds

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The Rat Cytokine array panel A (R&D system, Minneapolis, MN) was employed for the profiling of the chemokines and cytokines secreted upon CL and CSCL scaffolds implantation. This array allows the detection of 29 different molecules. We collected and quantified the proteins adsorbed on the scaffolds using the procedure described above (Bio-Rad). The results were obtained using the manufacturer’s instruction. The protein array images were scanned and pixel density analyzed by Molecular Imager ChemiDoc XRS System + Image Lab Software v.4.1 (BioRad). The experiments were performed in triplicate.

Corradetti B., Taraballi F., Corbo C., Cabrera F., Pandolfi L., Minardi S., Wang X., Van Eps J., Bauza G., Weiner B, & Tasciotti E. (2017). Immune tuning scaffold for the local induction of a pro-regenerative environment. Scientific Reports, 7, 17030.

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