V-PLEX, U-PLEX, and R-PLEX assays from the Meso Scale Discovery multispot assay system were used to quantify proteins in aorta tissue culture medium, BMDM culture medium, and plasma. Customized panels were used according to the manufacturer’s instructions. V-PLEX Panel 1 (K15048D) includes IL-1β, IL-4, IL-6, IL-10, IL-12, and CXCL1. V-PLEX Panel 2 (K15245D) includes CCL2, CCL3, CXCL2, and CXCL10. CCL5 (K152A2K-1), TGF-β Combo (K15242K-1) and MMP9 (B22ZG-2) were measured by U-PLEX assay. TIMP1 (F22YO-3) was measured by R-PLEX assay. MMP12 was measured by ELISA (ab213878, Abcam) according to the manufacturer’s instructions.
Multi spot assay system
Manufactured by Mesoscale
Sourced in United States
The MULTI-SPOT Assay System is a versatile laboratory instrument designed for high-throughput screening and quantification of multiple analytes in a single sample. The system utilizes a multi-array format to enable the simultaneous detection and measurement of various target molecules, such as proteins, peptides, and small molecules. The core function of the MULTI-SPOT Assay System is to provide a streamlined and efficient platform for researchers and scientists to conduct complex bioassays and immunoassays in a parallelized manner.
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8 protocols using multi spot assay system
1
Multispot Assay for Protein Quantification
2
Quantifying Metabolic Markers in Blood
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Blood samples were taken from the tail vein. Blood glucose and lactate levels were measured directly with Contour Next (Bayer) and Lactate Plus (Nova Biochemical) strips, respectively. Insulin and leptin levels were measured by a MULTI-SPOT Assay System (#K15124C, Meso Scale Discovery) according to the instructions from the manufacturer. This assay detects leptin and insulin in a multiplexed sandwich immunoassay. The sample and a solution containing labeled detection antibodies were added to a plate that was pre-coated with leptin and insulin capture antibodies on spatially distinct spots. Reading buffer was added and intensity of emitted light was measured on a QuickPlex SQ 120 (Meso Scale Discovery) to obtain a quantitative measure of leptin and insulin present in the sample.
3
Measuring Aβ Peptide Levels in APP-Overexpressing Cells
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SH-SY5Y cells overexpressing either Swedish-mutant APP or the isoforms APP695 or APP751 were seeded in 24-well plates (100,000 cells/well) and after one day they were washed twice with FBS-supplemented medium and incubated for 6 h with the inhibitor LY2811376 from Eli Lilly (Indianapolis, IN) or the different bioconjugates (SI, ANG-SI, ANG-PEG-SI, DHC-SI, TAT-SI, TRP-SI) at 5 μM. Supernatant from the tissue culture plates was then collected in low protein-binding tubes and analyzed by enzyme-linked immunosorbent assay with the Multi-Spot Assay System from Meso Scale Discovery (Rockville, MD) detecting human Aβ38, Aβ40 and Aβ42 peptides (#K15200E,) following manual’s instructions. The values obtained for the different conjugates varied in intensity (Aβ42 ~70,000 RU, Aβ40 ~400,000 RU and Aβ38 ~4,000 RU) and were normalized for the basal Aβ level detected in the supernatant of DMSO-treated cells, which was set as 100%.
4
Quantifying Secreted Amyloid Peptides
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Control and AD iNs were converted for 3 weeks and purified by FACS and re-plated on 8-well Geltrex-coated ibidi μ-slides on a density of 50K cells per well. After 4 weeks of conversion, 125 ul of 72 h-conditioned media were subjected to ELISA. Secreted Aβ40 and Aβ42 peptides were quantified by a sandwich immunoassay using the Meso Scale Discovery Multi-spot Assay System according to the manufacturer’s protocol, with the exception that incubation of detection antibody solution was performed overnight at 4°C.
5
Inflammatory Cytokine Profiling in Exercise
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Blood samples were taken at baseline and within 30 min post-exercise at the four consecutive exercise days. A venous blood sample was collected in a 10 mL EDTA vacutainer (Becton-Dickinson, NJ). The vacutainer was put on melting ice water and centrifuged at 1200 rcf for 15 min at 4 °C. Plasma was then transferred to polypropylene tubes and stored at -80 °C until analysis. IL-6, IL-8, IL-10, IL-1β and TNF-α concentrations were determined using the MesoScale Discovery (MSD) MULTI-SPOT Assay System [Proinflammatory Panel 1 (human) Kits, K15049D] according to the manufacturers' instructions. The lower detection limits varied per plate and were 0.136-0.432 (IL-6), 0.052-0.138 (IL-8), 0.039-0.213 (IL-10), 0.013-0.080 (IL-1β) and 0.066-0.229 (TNF-α) pg/ml. All values below these lower detection limits were considered as missing's. The percentages of missing values were 3.8% for IL-8 and TNF-α, 8.6% for IL-6 and IL-1β and 20.5% for IL-10. These values were imputed during statistical analysis. All standards for the calibration curve, control samples, and 10% of the plasma samples from participants were measured in duplicate. Accuracy and precision were evaluated by controls across multiple runs and multiple lots as described in the manufacturer's protocol.
6
Assessing Circulating miRNA Profiles
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According to standard protocols, the fasting venous blood samples were collected in BD Vacutainer® blood tubes. An earlier publication provided a thorough explanation of the sample collection and biochemical analysis methods [43 (link)]. Chemical-clinical analyses were done as part of routine laboratory testing, in a central laboratory (the University of Bremen, Centre for Biomolecular Interactions Bremen-CBIB). Serum samples stored at −80 °C were used to detect levels of hs-CRP, (using either single or MULTI-SPOT® Assay Systems, Meso Scale Discovery, Rockville, MD, USA).
Taking advantage of the qPCR array technology, we previously determined c-miRNA profiles in children and adolescents of the I.Family study [40 (link)]. Methods for miRNA extraction and screening from plasma samples have been previously published [35 (link),40 (link)]. Individual plasma samples were first tested for hemoglobin levels and hemolyzed samples were omitted from the analysis [35 (link)]. Different assays were performed in triplicate by using the miScript Primer Assays according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Relative miRNA levels were normalized using the endogenous spike-in Cel-miR-39 [35 (link)] employing the Data Assist v3.1 software package (Life Technologies, Thermo Fisher Scientific, Milan, Italy).
Taking advantage of the qPCR array technology, we previously determined c-miRNA profiles in children and adolescents of the I.Family study [40 (link)]. Methods for miRNA extraction and screening from plasma samples have been previously published [35 (link),40 (link)]. Individual plasma samples were first tested for hemoglobin levels and hemolyzed samples were omitted from the analysis [35 (link)]. Different assays were performed in triplicate by using the miScript Primer Assays according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Relative miRNA levels were normalized using the endogenous spike-in Cel-miR-39 [35 (link)] employing the Data Assist v3.1 software package (Life Technologies, Thermo Fisher Scientific, Milan, Italy).
7
Standardized Biomarker Sampling and Analysis
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The fasting venous blood draws were collected in BD Vacutainer® blood tubes according to standardized operative procedures. A complete description of the sample collection and investigative procedures has been earlier published [34 (link)]. Clinical chemistry tests were determined as part of routine laboratory testing, in a central laboratory (University of Bremen, Centre for Biomolecular Interactions Bremen—CBIB). Serum samples stored at −80 °C were used to detect levels of CRP, Interleukin-1 Receptor Antagonist (IL1-Ra), IL-6, IL-8, Interleukin-15 (IL-15), and TNF-α using an electrochemiluminescent multiplex assay (using either single or MULTI-SPOT® Assay Systems, Meso Scale Discovery).
8
Comprehensive Inflammatory Biomarker Profiling
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Serum samples stored at -80°C were used to detect levels of C-reactive protein (CRP), interleukin-1 receptor antagonist (IL-1Ra), IL-6, 8, 15, interferon gamma inducible protein (IP-10), TNF-α, adiponectin and leptin were measured at T 0 and T 3 , by ELISA using electrochemiluminescent multiplex assay (using either single or MULTI-SPOT® Assay Systems, Meso Scale Discovery). The choice of inflammatory markers were based on their role in endothelial function via either direct or indirect mechanisms such as reducing nitric oxide production and stimulating inflammation-oxidative stress pathways. IL-6, IL-8, TNF-α, IP-10, IL-15 and IL-1Ra were run together on a 6-plex assay, insulin and leptin run together on a 2-plex assay, whereas adiponectin, and CRP on single-plex assays each. The combination of markers for the assays were decided based on the feasibility of combinations with the help of MSD customer support.
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