The plasma samples were centrifuged at 4 °C at 3000 rpm for 10 minutes, the supernatant was collected and diluted 2-fold for further analysis. Concentrations of IL-1β, TNF-α, IL-6 and IFNγ were determined using the Proinflammatory Panel 1 (mouse) V-PLEXTM Kit and QuickPlex SQ 120 (Mesoscale Discovery, Rockville, MD) according to manufacturer’s protocols. Cytokines were expressed as ng/ml plasma.
Mouse 5 plex proinflammatory panel 1 kit
Manufactured by Mesoscale
Sourced in United States
The Mouse V-PLEX Proinflammatory Panel 1 Kit is a laboratory equipment product designed for the quantitative measurement of multiple proinflammatory analytes in mouse biological samples. The kit utilizes the Meso Scale Discovery (MSD) multi-array technology to provide simultaneous detection of multiple analytes from a single sample.
Automatically generated - may contain errors
Lab products found in correlation
Quickplex sq 120 apparatus, by Mesoscale (3 mentions)
Quickplex sq 120, by Mesoscale (2 mentions)
Duoset elisa, by R&D Systems (2 mentions)
Mouse leptin kit, by Mesoscale (2 mentions)
Mouse il 1ra elisa kit, by Thermo Fisher Scientific (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Mouse duoset elisa kit, by R&D Systems (1 mentions)
Mouse creatine kinase mb elisa kit, by Novus Biologicals (1 mentions)
Duoset elisa kit, by R&D Systems (1 mentions)
Free glycerol reagent, by Merck Group (1 mentions)
Triglyceride reagent, by Merck Group (1 mentions)
9 protocols using mouse 5 plex proinflammatory panel 1 kit
1
Cytokine Profiling in Mouse Plasma
2
Quantifying Intestinal Inflammation Cytokines
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The small intestine mucosa scrapings (50~100 mg) were mixed with 400 μL lysis buffer (150 mM NaCl, 20 mM Tris, 1 mM EDTA, 1% Triton X-100, pH 7.5) and phosphatase and protease inhibitors (Sigma, St. Louis, MO) were added to inhibit degradation. Samples were homogenized and then centrifuged at 4℃ at 12000 rpm for 30min, the protein fraction was collected, and the total protein concentrations were measured in the extract using the Bradford assay. Concentrations of IL-1β, TNF-α, IL-6 and IFNγ were determined using the Proinflammatory Panel 1 (mouse) V-PLEXTM Kit and QuickPlex SQ 120 (Mesoscale Discovery, Rockville, MD) according to manufacturer's protocols. Cytokines are expressed as ng/mg protein.
3
Cytokine Profiling in Mouse Colon
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Snap frozen colonic tissue was homogenized in PBS containing 0.05% Tween 20. Homogenates were centrifuged at 9600 × g for 10 minutes. IFN-γ, IL-1β, IL-10, IL-12 p70, IL-2, IL-4, IL-5, KC/GRO (keratinocyte chemoattractant/human growth-regulated oncogene), IL-6, and TNF-α were evaluated using the Proinflammatory Panel 1 V-PLEX Mouse Kit (Meso Scale Discovery, Rockville, MD) as per manufacturer’s protocol. TGF-β was measured using ELISA duo set (R&D Systems, Inc., Minneapolis, MN). Cytokine levels were corrected for tissue weight.
4
Intestinal Cytokine and MPO Profiling
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Snap frozen peri-anastomotic colon and ileum were homogenized in PBS containing 0.05% Tween 20. Homogenates were centrifuged at 10 000 rpm for 10 minutes. Cytokine levels were corrected for dry tissue weight. IFN-γ, IL-1β, IL-10, IL-12 p70, IL-2, IL-4, IL-5, IL-6, KC/GRO, and TNF-α were evaluated using the Proinflammatory Panel 1 V-PLEX Mouse Kit (Meso Scale Discovery, Rockville, MD) as per manufacturer’s protocol. IL-10, IL-6, TNF-α, and TGF-β also measured using the ELISA duo sets (R&D Systems, Inc., Minneapolis, MN). Myeloperoxidase (MPO) was measured using the MPO, Mouse, ELISA kit (Hycult Biotech Inc., Plymouth Meeting, PA).
5
Cytokine Quantification from Plasma and Tissue
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For blood analysis, plasmas were diluted twice and analysis following manufacturer’s instructions using the mouse V-PLEX Proinflammatory Panel 1 Kit (Meso Scale Discovery, # K15048D) on a QuickPlex SQ 120 apparatus (Meso Scale Discovery).
For tissue analysis, proteins were extracted from frozen organs using an ULTRA TURRAX T25 (Ika, Germany) and lysis buffer (25 mM Tris-Cl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P40 and protease inhibitors (Roche Diagnostics, Meylan, France)). Protein concentration was evaluated by BCA assay (Sigma-Aldrich Chimie, Saint-Quentin Fallavier, France). 10 µg proteins were used to evaluate cytokine concentration using the same kit and apparatus as those used for blood cytokine analysis.
For tissue analysis, proteins were extracted from frozen organs using an ULTRA TURRAX T25 (Ika, Germany) and lysis buffer (25 mM Tris-Cl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% Nonidet P40 and protease inhibitors (Roche Diagnostics, Meylan, France)). Protein concentration was evaluated by BCA assay (Sigma-Aldrich Chimie, Saint-Quentin Fallavier, France). 10 µg proteins were used to evaluate cytokine concentration using the same kit and apparatus as those used for blood cytokine analysis.
6
Quantifying Inflammatory Markers in Mice
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IFNβ and IL-6 concentrations in organ homogenates were analyzed using mouse DuoSet ELISA kits (R&D Systems). Quantification of serum IL-6, IL-8, IL-1β, and TNFα was performed using a mouse V-PLEX Proinflammatory Panel 1 kit (Meso Scale Diagnostics) and was analyzed by the OSU Center for Clinical and Translational Science. Creatine kinase quantification was performed using the Mouse Creatine Kinase MB ELISA Kit (Novus Biologicals).
7
Quantification of Inflammatory Markers in Murine Lung Tissue
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IL‐6 ELISAs were performed on fluid from lung homogenates using an R&D Systems Duoset ELISA kit (catalog # DY406) according to the manufacturer's instructions. Quantification of serum IL‐1β and TNFα was performed using a mouse V‐PLEX Proinflammatory Panel 1 kit (Meso Scale Diagnostics) and was analyzed by the OSU Center for Clinical and Translational Science. For lung histology, lung tissue samples were fixed in formalin for 7 days at 4°C, followed by embedding in paraffin. For H&E and anti‐CD45 staining, lungs were sectioned, stained, and imaged by the OSU Comparative Pathology and Mouse Phenotyping Shared Resource. Quantification of these images was performed using ImageJ software and the color deconvolution method. Staining for SARS‐CoV‐2 antigen was performed and imaged by Histowiz (Histowiz.com , Brooklyn, NY, USA).
8
Adipose Tissue Secretome Analysis
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For blood analysis, freshly prepared plasmas were diluted twice before analysis. For tissue analysis, freshly sampled WAT and BAT were washed in PBS, weighed and incubated in free DMEM (Dulbecco’s modified Eagle medium) for 2 hours at 37°C. Media were kept for various analyses of secreted proteins.
Leptin and cytokines were assayed using “Mouse Leptin Kit” (Meso Scale Discovery, # K152BYC) and “mouse V-PLEX Proinflammatory Panel 1 Kit” (Meso Scale Discovery, # K15048D) respectively, on a QuickPlex SQ 120 apparatus (Meso Scale Discovery) following manufacturer’s instructions. IL-1RA levels were assayed using mouse IL-1RA Elisa kit (#EMIL1RN) from Thermo Fisher Scientific (Courtaboeuf, France). Glycerol and triglycerides determinations were performed using dedicated kit (Free Glycerol reagent and Triglyceride reagent, Sigma Aldrich).
Leptin and cytokines were assayed using “Mouse Leptin Kit” (Meso Scale Discovery, # K152BYC) and “mouse V-PLEX Proinflammatory Panel 1 Kit” (Meso Scale Discovery, # K15048D) respectively, on a QuickPlex SQ 120 apparatus (Meso Scale Discovery) following manufacturer’s instructions. IL-1RA levels were assayed using mouse IL-1RA Elisa kit (#EMIL1RN) from Thermo Fisher Scientific (Courtaboeuf, France). Glycerol and triglycerides determinations were performed using dedicated kit (Free Glycerol reagent and Triglyceride reagent, Sigma Aldrich).
9
Adipose Tissue Secretome Analysis
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For blood analysis, freshly prepared plasma was diluted twice before analysis. For tissue analysis, freshly sampled WAT and BAT were washed in PBS, weighed and incubated in free Dulbecco's modified Eagle's medium for 2 hours at 37°C. The media were preserved for analysis of various secreted proteins.
Leptin was assayed using a mouse leptin kit (Meso Scale Discovery, # K152BYC), and cytokines were measured using a mouse V-PLEX pro-inflammatory panel 1 kit (# K15048D) according to the manufacturer's instructions using a QuickPlex SQ 120 apparatus (Meso Scale Discovery, Rockville, Maryland, USA). IL-1RA levels were assayed using a mouse IL-1RA
ELISA kit (#EMIL1RN) from Thermo Fisher Scientific (Courtaboeuf, France). Glycerol and triglyceride determinations were performed using a dedicated kit (free glycerol reagent and triglyceride reagent, Sigma Aldrich).
Leptin was assayed using a mouse leptin kit (Meso Scale Discovery, # K152BYC), and cytokines were measured using a mouse V-PLEX pro-inflammatory panel 1 kit (# K15048D) according to the manufacturer's instructions using a QuickPlex SQ 120 apparatus (Meso Scale Discovery, Rockville, Maryland, USA). IL-1RA levels were assayed using a mouse IL-1RA
ELISA kit (#EMIL1RN) from Thermo Fisher Scientific (Courtaboeuf, France). Glycerol and triglyceride determinations were performed using a dedicated kit (free glycerol reagent and triglyceride reagent, Sigma Aldrich).
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