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Mouse proinflammatory 7 plex ultra sensitive kit

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The Mouse ProInflammatory 7-Plex Ultra-Sensitive Kit is a laboratory equipment product designed for the quantitative measurement of seven mouse pro-inflammatory cytokines in a single sample. The kit utilizes a multiplexed immunoassay technology to detect and quantify the target analytes.

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12 protocols using mouse proinflammatory 7 plex ultra sensitive kit

1

Kidney and Lung Injury Assessment

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Serum creatinine and BUN were measured using a VetAce autoanalyzer (Alfa Wassermann, West Caldwell, NJ). Lung MPO activity was determined on ¼ lung section as previously described 29 (link). Blood pH was measured using a blood gas analyzer (Siemens RapidLab 248). ATN scores and other markers of kidney injury were determined as previously described 30 (link) and as detailed in supplemental methods. Serum, dialysate, and peritoneal cell cytokines were determined using a mouse proinflammatory 7-plex ultra-sensitive kit (MesoScale Discovery, Gaithersburg, MD, USA) per manufacturer’s instructions. Serum IL-6 and CXCL1 were measured by ELISA (R and D), per manufacturer’s instructions. Other lung injury assessments and flow cytometry for myeloid derived cells were performed as detailed in supplemental methods.
Altmann C., Ahuja N., Kiekhaefer C.M., Hernando A.A., Okamura K., Bhargava R., Duplantis J., Kirkbride-Romero L.A., Huckles J., Fox B.M., Kahn K., Soranno D., Gil H.W., Teitelbaum I, & Faubel S. (2017). Early peritoneal dialysis reduces lung inflammation in mice with ischemic acute kidney injury. Kidney international, 92(2), 365-376.
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2

Murine Inflammatory Cytokine Profiling

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An additional seven mice were used as for cytokine measurements. Post-mortem cardiac puncture blood samples were obtained. Serum interferon gamma (IFNγ), interleukin-10 (IL-10), interleukin-12p70 (IL-12p70), interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNFα) and chemokine (C-X-C motif) ligand 1 (CXCL 1) levels were measured using the Mouse ProInflammatory 7-Plex Ultra-Sensitive Kit (Meso Scale Discovery, Gaithersburg, MD, USA) according to the manufacturer’s instructions. Assay sensitivities were 0.38, 11, 35, 0.75, 4.5, 0.85, and 3.3 pg/ml, respectively. The intra-assay and inter-assay variations were less than 10% for all markers. Serum insulin-like growth factor 1 (IGF-1) levels were measured using a kit (IDS, Fountain Hills, AZ, USA) according to the manufacturer’s instructions. Assay sensitivity was 2.8 ng/ml. Intra- and inter-assay variation were 5.8% and 7.8%, respectively. All measurements were performed in duplicate.
Yagi M., Arentsen L., Shanley R.M., Rosen C.J., Kidder L.S., Sharkey L.C., Yee D., Koizumi M., Ogawa K, & Hui S.K. (2014). A Dual Isotope Hybrid Whole Body Micro-PET/CT System Reveals Functional Heterogeneity and Early Local and Systemic Changes Following Targeted Radiation to the Murine Caudal Skeleton. Calcified tissue international, 94(5), 544-552.
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3

Assessing Lung Inflammation via BALF

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Following euthanasia, the lungs were lavaged twice with 0.5 mL phosphate-buffered saline passed through a 19-gauge cannula anchored in the trachea. Pooled broncho-alveolar lavage fluid (BALF) was immediately placed on ice. Three-hundred cell differential counts were performed on modified Wright’s-stained cytocentrifuge slide preparations of 400 μL aliquots of raw BALF. Levels of the BALF cytokines IFN-γ, IL-1β, IL-6, IL-10, IL-12p70, KC/GRO/CINC, and TNF-α were assayed with the Mouse ProInflammatory 7-Plex Ultra-Sensitive Kit (Meso Scale Diagnostics, Rockville, Maryland). The lower limit of detection for all the cytokines ranged from 0.38–35 pg/mL. This assay was performed as specified by the manufacturer.
Noël A., Xiao R., Perveen Z., Zaman H.M., Rouse R.L., Paulsen D.B, & Penn A.L. (2016). Incomplete lung recovery following sub-acute inhalation of combustion-derived ultrafine particles in mice. Particle and Fibre Toxicology, 13, 10.
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4

Cytokine and Signaling Pathway Analysis

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Cytokines were measured using the Mouse Proinflammatory 7-Plex Ultra-Sensitive kit (Meso Scale Discovery). Serum IgM was measure by ELISA (eBioscience). To examine protein expression in tissues, lysates were run on SDS-PAGE and blotted onto nitrocellulose membranes (Invitrogen). Blots were probed for β-actin (Sigma), RIP1 (BD Biosciences) and RIP3 (Imgenex). For TNF activation experiments, BMDM were treated with 50ng/ml of TNF (R&D systems) and lysates were separated by SDS-PAGE and blotted onto a nitrocellulose membrane (Invitrogen). Blots were probed for IκB, phospho-IκB, tubulin, phospho-p38, total p38, phospho-JNK and total JNK (Cell Signaling), phospho-ERK and total ERK (Santa Cruz).
Berger S.B., Kasparcova V., Hoffman S., Swift B., Dare L., Schaeffer M., Capriotti C., Cook M., Finger J., Hughes-Earle A., Harris P.A., Kaiser W.J., Mocarski E.S., Bertin J, & Gough P.J. (2014). RIP1 kinase activity is dispensable for normal development but is a key regulator of inflammation in SHARPIN-deficient mice. Journal of immunology (Baltimore, Md. : 1950), 192(12), 5476-5480.
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5

Evaluating Pulmonary Barrier Integrity

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Analysis of endothelial–epithelial barrier permeability was performed by measuring mouse serum albumin in recovered BAL fluid by ELISA according to the manufacturer’s instructions (Bethyl Laboratories). Coagulation activation was determined by measuring thrombin–antithrombin complexes (TAT) in BAL fluid by ELISA (EIAab). Cytokines (IFN-γ, IL-1β, IL-6, IL-10, and TNF) and chemokine (KC/Gro) were measured with the mouse proinflammatory 7-plex ultra-sensitive kit (Meso Scale Discovery, Rockville, MD) according to the manufacturer’s protocol. The chemokine CCL2 (MCP-1) was measured by ELISA (R&D Systems Europe) or the mouse CCL2 ultra-sensitive kit (Meso Scale Discovery) according to the manufacturer’s protocol, and CCL7 (MCP-3) was measured by ELISA (PromoKine) according to the manufacturer’s protocol.
José R.J., Williams A.E., Mercer P.F., Sulikowski M.G., Brown J.S, & Chambers R.C. (2015). Regulation of Neutrophilic Inflammation by Proteinase-Activated Receptor 1 during Bacterial Pulmonary Infection. The Journal of Immunology Author Choice, 194(12), 6024-6034.
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6

Multiplex Cytokine Quantification in Serum

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The concentrations (pg/mL) of cytokines were measured in serum in duplicates using a commercial kit: Mouse ProInflammatory 7-Plex Ultra-Sensitive Kit (Meso scale Discovery: Rockville). The measurements were performed at the Center of Inflammation and Metabolism, Copenhagen University Hospital, Rigshospitalet in accordance with manufacturer's instructions. The coefficient of variation was at low, medium and high concentrations IL1b: 8.4%; 1.8% and 3.2% (highest at lowest concentration), IL-12: 8.1%; 2.3% and 4.4%, IFN-y: 7.3%; 1.0% and 2.9%, IL-6: 2.8%; 6.8% and 4.0%; KC/GRO: 7.8%; 5.7% and 2.8%; IL-10: 4.3%; 1.1% and 5.8% and TNFα: 9.0%; 3.4% and 2.6%.
The lower limits of detection (LLOD) were: IL1b: 0.75 pg/mL; IL-12: 35 pg/mL; IFN-y: 0.38 pg/mL; IL-6: 4.5 pg/mL; KC/GRO: 3.3 pg/mL; IL-10: 11 pg/mL and TNFα: 0.85 pg/mL.
Kvist T.M., Syberg S., Petersen S., Ding M., Jørgensen N.R, & Schwarz P. (2015). The role of the P2X7 receptor on bone loss in a mouse model of inflammation-mediated osteoporosis. Bone Reports, 7, 145-151.
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7

Plasma biomarker analysis protocol

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The trunk blood was collected in EDTA and centrifuged at 2000g for 15 min at 4 °C. The plasma was collected and stored at − 80 °C until measurements for LCN2 (Lipocalin-2/NGAL Quantikine ELISA Kit, R&D Systems), IL-1, IL-6 (Mouse Proinflammatory 7-Plex Ultra-Sensitive Kit, Meso Scale Discovery immunoassays), triglycerides ELISA (Charles River Lab), total cholesterol (Cholesterol Quantitation Kit, Sigma-Aldrich), and insulin and leptin (Mouse Metabolic Kit (Multi-spot Assay System, Meso Scale Discovery). Plates were processed in a SECTOR® Imager 6000 plate reader (Meso Scale Diagnostics, LLC). Data acquired using the Discovery Workbench software (v4.0; Meso Scale Diagnostics, LLC).
De Sousa Rodrigues M.E., Houser M.C., Walker D.I., Jones D.P., Chang J., Barnum C.J, & Tansey M.G. (2019). Targeting soluble tumor necrosis factor as a potential intervention to lower risk for late-onset Alzheimer’s disease associated with obesity, metabolic syndrome, and type 2 diabetes. Alzheimer's Research & Therapy, 12, 1.
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8

Multiplex Serum Cytokine Analysis

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Serum cytokines were analyzed on a Mouse ProInflammatory 7‐Plex Ultra‐Sensitive Kit (cat # K15012C; MesoScale Discovery, Gaithersburg, MD),
Andres‐Hernando A., Altmann C., Bhargava R., Okamura K., Bacalja J., Hunter B., Ahuja N., Soranno D, & Faubel S. (2014). Prolonged acute kidney injury exacerbates lung inflammation at 7 days post‐acute kidney injury. Physiological Reports, 2(7), e12084.
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9

Comprehensive Inflammatory and Metabolic Profiling

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The inflammatory cytokines (interferon γ, tumor necrosis factor α, interleukin 1β [IL‐1β], IL‐6, IL‐12, keratinocyte chemoattractant/human growth‐regulated oncogene, and IL‐10) in plasma, kidney, and BMDM culture medium were detected using a Mouse Proinflammatory 7‐Plex Ultra‐Sensitive Kit (MesoScale Discovery). Kidneys were homogenized in Tris‐lysis buffer (150 mmol/L NaCl, 20 mmol/L Tris, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X‐100, pH 7.5) with 1% BSA (Sigma‐Aldrich) using a Bullet Blender. Protein levels in the supernatant were quantified with a Bradford protein assay (Bio‐Rad Laboratories), and 50 μg protein was loaded for analysis. The plasma metabolic markers (total glucagon‐like peptide 1, glucagon, insulin, and leptin) were detected with MSD MULTI‐SPOT 96‐well, 4‐spot prototype Mouse Metabolic Kit (MesoScale Discovery), and plasma adiponectin levels were detected with the Adiponectin Mouse ELISA kit (ab108785; Abcam). Urine samples, obtained from metabolic cages, were analyzed for the oxidative stress marker 8‐hydroxy‐2‐deoxyguanosine (8‐OH‐dG) using the DNA/RNA Oxidative Damage ELISA Kit (589320; Cayman). Circulating levels of angiotensin II (Ang II) were analyzed by the Angiotensin II EIA Kit (RAB0010; Sigma‐Aldrich).
Yang T., Zollbrecht C., Winerdal M.E., Zhuge Z., Zhang X., Terrando N., Checa A., Sällström J., Wheelock C.E., Winqvist O., Harris R.A., Larsson E., Persson A.E., Fredholm B.B, & Carlström M. (2016). Genetic Abrogation of Adenosine A3 Receptor Prevents Uninephrectomy and High Salt–Induced Hypertension. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 5(7), e003868.
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10

Inflammatory Cytokine Profiling in Malaria

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Serum from uninfected and P. berghei ANKA infected WT and ApoE−/− mice was isolated as outlined above and serum concentrations of IFN-γ, IL-1β, IL-6, IL-10, IL-12p70, KC/GRO/CINC, and TNF-α were quantified using the Mouse ProInflammatory 7-Plex Ultra-Sensitive Kit (Meso Scale, Gaithersburg, MD), according to the manufacturer’s protocol. The plates were analyzed by a SECTOR Imager 2400 plate reader (Meso Scale).
Kassa F.A., Van Den Ham K., Rainone A., Fournier S., Boilard E, & Olivier M. (2016). Absence of apolipoprotein E protects mice from cerebral malaria. Scientific Reports, 6, 33615.
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