Blood was collected by heart puncture following sacrifice and centrifuged at 1500 × g, at 4 °C for 15 min to isolate the plasma, which was stored at −80 °C until analysis. Levels of proinflammatory proteins, cytokines, and Th17 mediators were analyzed using V‐PLEX Mouse Cytokine 29‐Plex Kit (Meso Scale Diagnostics, Rockville, MD) and Discovery Workbench 4.0 software according to manufacturer’s specifications.
V plex mouse cytokine 29 plex kit
Manufactured by Mesoscale
Sourced in United States
The V-Plex Mouse Cytokine 29-plex kit is a multiplex immunoassay panel designed to detect and quantify the levels of 29 different cytokines in mouse samples. The kit utilizes Mesoscale's proprietary electrochemiluminescence detection technology to provide accurate and reliable measurements of these immunomodulatory proteins.
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7 protocols using v plex mouse cytokine 29 plex kit
1
Plasma Cytokine Profiling in Mice
2
Multiplex Cytokine Profiling in Mice
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Blood was sampled by cardiac puncture from anaesthetized mice which were subsequently euthanized. The blood was centrifuged at 8000 g for 8 min and plasma was used to measure cytokines with a multiplex V-PLEX mouse cytokine 29-Plex kit (Mesoscale Discovery, Rockville, MD) according to manufacturer’s instructions. IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, CXCL1, IL-10, IL-12p70, IL-15, IL17A/F, IL-27p28/IL30, IL-33, CXCL10, CCL2, CCL3, CXCL2, TNF-α, CCL20, IL-22, IL-23, IL-17C, IL-31, IL-21, IL-17F, IL-16, IL-17A, IL-17E/IL-25 were included in the kit. IL-6, IL-17F, IL-21, IL-23, IL-31, IL-9, IL-17A/F, IL-12p70, IL-4 were below detection range. Results were read and analyzed on a MESO QuickPlex SQ120.
3
Multiplex Inflammation Protein Profiling
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Blood was obtained by cardiac puncture immediately after sacrifice using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Plasma was separated by centrifugation, and stored at −80 °C until use. Plasma inflammatory protein measurements were obtained by utilizing the Meso Scale Discovery V-Plex Mouse Cytokine 29-plex kit (#K15267D, Rockville, MD, USA). Meso Scale Discovery V-plex plates are tightly validated by the manufacturer to avoid antigenic cross-reactivity. The following inflammatory proteins were measured: IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-15, IL-16, IL-17A, IL-17C, IL-17F, IL-17A/F, IL-17E/IL-25, IL-21, IL-22, IL-23, IL-27p28/IL-30, IL-31, IL-33, CXCL10, CXCL1, CCL2, CCL3, CXCL2, CCL20, TNF-α. All experiments were conducted per the manufacturer’s instructions, with samples run on Meso Scale Discovery Quickplex SQ 120 and analyzed using the Mesoscale Discovery Workbench software.
4
Multiplex Cytokine Profiling in Mice
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Concentrations of a panel of 29 murine cytokines and chemokines were analyzed using the V-PLEX Mouse Cytokine 29-Plex kit on the Meso Scale Discovery (MSD) platform (Meso Scale Diagnostics, Rockville, MD, USA), according to the manufacturer’s protocol. Data was analyzed using Discovery Workbench 4.0 software (Meso Scale Diagnostics). Serial dilutions of specific recombinant cytokines and chemokines were used to establish a standard curve to determine concentrations of each of the analytes measured.
5
Multiplex Inflammation Protein Profiling
6
Cytokine Profiling in Murine BALF and Lung
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BALF samples were centrifuged (150xg for 10 min) to obtain cell-free supernatant. BALF (50 µL) was used for cytokine assessment. Lung tissue specimen from the right lung middle lobe was collected in Tissue Protein Extraction Reagent T-Per (Pierce; Thermofisher Scientific, Rockford, IL, USA) containing Protease Inhibitor Cocktail (Sigma Aldrich, Oakville, ON, Canada). Tissue was homogenized on ice using the Cole-Parmer LabGEN 125 Homogenizer (Canada Inc, Montreal, QC, Canada). Homogenates were centrifuged (10,000 x g) to obtain tissue lysate. Protein amount in the tissue lysate was quantified with bicinchoninic acid (BCA) Protein Assay (Pierce). Total protein (50 µg) was used from each lung tissue lysate for cytokine evaluation. BALF and lung tissue lysates were aliquoted and stored at -20 °C until used. Abundance of a panel of murine cytokines and chemokines was measured in BALF and lung tissue lysates using the V-plex Mouse Cytokine 29-Plex Kit and the multiplex Meso Scale Discovery (MSD) platform (Meso Scale Discovery, Rockville, MD, USA), as per the manufacturer’s instructions. Data was analyzed using the Discovery Workbench 4.0 software (Meso Scale Discovery).
7
Cytokine Profiling via Multiplex Assay
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Circulating cytokines were measured as previously reported (33) . In brief, blood was obtained by cardiac puncture immediately following sacrifice with anti-coagulation maintained through addition of ethylenediaminetetraacetic acid (EDTA). Plasma was separated from whole blood by centrifugation and stored at -80°C until assay. Cytokine concentration was assessed by Meso Scale Discovery V-Plex Mouse Cytokine 29-plex kit (#K15267D, Rockville, MD, USA). All experiments were conducted per manufacturer's instructions and quantified on a Meso Scale Discovery Quickplex SQ 120, with analyses conducted using Mesoscale Discovery Workbench software (Rockville, MD, USA).
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