To study KD effects on inflammatory cytokines, 12 weeks old male mice were randomly assigned to either chow, KD or HFD for 3 days and 16 weeks. Mice were fasted for 6 h and injected glucose 30 min, or without glucose injections before tissue and blood sampling. Circulating IL-6 concentration, IL-6 in liver, mesenteric fat, and ingWAT lysate were measured by using Mouse IL-6 ELISA Kit (RayBiotech, #ELM-IL6, Luzern, Switzerland). Liver cholesterol was measured by Cobas Roche (Hitachi Kit #11877771, Roche Diagnostics International). Plasma cytokine Interferon gamma (IFN-γ), interleukin 1 beta (IL-1β), interleukin 2 (IL-2), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin-12 (IL-12p70), tumor necrosis factor alpha (TNF-α), and keratinocyte chemoattractant (KC)/growth-regulated oncogene (GRO) chemokines and pro-inflammatory chemokines were measured using the MSD technology (Meso Scale Discovery, Gaithersburg, MD, USA). All analyses were carried out according to manufacturer's protocols.
Msd technology
Manufactured by Mesoscale
Sourced in United States
MSD technology is a sensitive and precise analytical technique used for the detection and quantification of a wide range of molecules, including proteins, peptides, and small molecules. The core function of this technology is to perform mass spectrometry analysis, providing accurate measurements of the molecular mass and abundance of target analytes in a sample.
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Lab products found in correlation
Mouse il 6 elisa kit, by RayBiotech (1 mentions)
Cobas mira, by Roche (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Mtesr plus, by Merck Group (1 mentions)
Meso quickplex sq 120 instrument, by Mesoscale (1 mentions)
Phosphoramidon, by Merck Group (1 mentions)
V plex aβ peptide panel 1 6e10 kit, by Mesoscale (1 mentions)
Prednisolone, by Merck Group (1 mentions)
Balb c mice, by Charles River Laboratories (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
8 protocols using msd technology
1
Ketogenic Diet Alters Inflammatory Cytokines
2
IFN-γ Production in PBMC Assay
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Cell culture supernatants were used to measure IFN‐γ production using MSD technology (Meso Scale Discovery, Gaithersburg MD). MSD assays were carried out following the manufacturer’s instructions. Briefly, PBMC were stimulated with LVS, and the supernatants harvested after 96 h and kept at −70°C until assayed. Based on linearity, the levels of sensitivity for IFN‐γ ranged from 0.47 to 2.91 pg mL−1. Responses were scored positive if (1) cytokine production was 2‐fold or higher than media cultures at a particular time point, and (2) the fold values (Schu‐S4 divided by media) at a specific point of time after immunisation were 2‐fold or higher than those at Day 0 (baseline) in at least one post‐vaccination visit.
3
Metabolic Biomarkers in Mouse Model
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Individual mouse body weights were monitored every week. Mice were fasted overnight for blood sampling. ASK1 inhibitor‐treated mice were fasted for 5 h before blood sampling. Free fatty acid (FFA), glycerol, triglyceride (TG), total cholesterol (TC) alanine transaminase (ALT), and aspartate aminotransferase (AST) levels were measured by Roche Cobas Mira (Sawgrass Drive, Bellport, NY, USA). The commercial kits were obtained from Diatools AG (Villmergen, Switzerland). Insulin was determined as previously described (Konrad et al, 2007 ). Plasma cytokine tumor necrosis factor alpha (TNF‐α), interleukin 10 (IL‐10), interleukin 6 (IL‐6), and keratinocyte chemoattractant (KC/CXCL1) were measured using MSD technology (Meso Scale Discovery, Gaithersburg, MD, USA). All analyses were carried out according to manufacturer's protocols.
4
Serum Cytokine Profiling Using MSD
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At the initial visit cytokines (IL-15, IL-7, IFN-γ, IL-10, IL-12, IL-13, IL1-beta, IL-2, IL-4, IL-6, IL-8, TNF-alpha and granulocyte macrophage colony stimulating factor (GM-CSF)) were measured in serum by MSD technology (Meso Scale Discovery, MD, USA) as per established protocol.
5
Measuring Interferon Subtypes in Autoimmune Disorders
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Serum IFN-α was measured using the digital Simoa platform as described.12 (link) Monoclonal antibodies (specifically for all IFN-α subtypes) were isolated from autoimmune polyendocrinopathy candidiasis ecto-dermal dystrophy patients13 (link) and provided to D. Duffy by Immunoqure under a material transfer agreement. IFN-β, IFN-γ and IFN-λ1/IL29 (referred to hereafter as IFN-λ) were measured by MSD technology (Meso Scale Discovery, MD, USA) as per manufacturers’ instructions.
6
Quantification of Aβ Peptides from hiPSC Conditioned Media
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hiPSCs were plated on matrigel pre-coated 12-well plates in triplicate. Two days later, medium was replaced by 500µL of mTeSR Plus supplemented with phosphoramidon (10 µM, Sigma-Aldrich). Twenty-four hours later, conditioned medium was collected in each well and centrifuged at 1000×g for 5 min. The supernatant was supplemented with a cocktail of protease inhibitors (Sigma-Aldrich) and frozen at − 80 °C until use. The quantification of Aβ peptides was performed using the MSD technology (M eso S cale D iagnostics, Rockville, MD, USA) with the V-PLEX Aβ Peptide Panel 1 (6E10) kit according to manufacturer’s instructions. Conditioned media were diluted 1:2 in Diluent 35 and analyzed in duplicate. The plate was analyzed using a MESO QuickPlex SQ 120 instrument (Meso Scale Diagnostics). Aβ quantities were normalized to the total protein amount measured in each sample.
7
Modulation of Airway Inflammation in HDM-Induced Asthma
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Balb/c mice (Charles River) were purchased from Charles River. The animals were housed under conventional conditions in isolated ventilated cages with free access to water and food. All experiments performed were approved by authorities for the care and use of experimental animals (Regierungspräsidium Tübingen, TVV15‐017). Mice were either treated sub cutaneous (s.c.) with CFA (Complete Freund's Adjuvant; Biomol) alone (control groups; per group n = 4) or with additional 100μg HDM (Greer)/ animal at day 0 (HDM treated groups, per group n = 8). At day 4, 1.5 × 1011 virus genomes of stuffer virus (negative control) or FKBP51‐AAVs were administered intra tracheal (i.t.) to the mice. AAV‐untreated mice received PBS i.t. as negative control. On day 14 and 15 HDM sensitized mice were re‐stimulated with 25μg HDM i.t. On the same days steroid‐treated mice got either 5 or 10 mg/kg prednisolone (Sigma‐Aldrich) b.i.d. via oral administration. Mice were sacrificed on day 16 measuring BALF neutrophils with a Sysmex XT‐1800i‐ device and BALF cytokines using the MSD technology (Meso Scale Discovery). BALF were generated by rinsing the lungs two times with PBS containing 1x cOmplete™ Protease Inhibitor Cocktail (Roche).
8
Reproducibility of Olink Proteomics
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We subsequently performed additional proteomics profiling using an MSD technology (MesoScale Discovery, Rockville, MD, USA) in 19 individuals from the study cohort to determine the reproducibility of our olink-based results. MSD procedure follows that of a sandwich ELISA, with any analytes of interest captured on the electrode being detected with an analyte specific ruthenium-conjugated secondary antibody. Values were presented as pg/ml on a log2 scale.
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