The cell culture supernatant was removed and stored at -70 °C and, later, destined for measuring cytokines by the Luminex xMAP® immunoassay methodology using the ProcartaPlex Immunoassay kit (Affymetrix eBioscience) according to the manufacturer’s instructions. The following cytokines were measured: GM-CSF; TNF-α; IL-1β; IL-6; IL-10; IL-12; IL-18; IL-23 and IL-27.
Procartaplex immunoassay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ProcartaPlex immunoassay kit is a multiplex assay platform designed to detect and quantify multiple analytes in a single sample. It provides a convenient and efficient way to measure a variety of protein targets in different sample types, such as cell culture supernatants, tissue homogenates, and biological fluids.
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Lab products found in correlation
Luminex xmap, by Thermo Fisher Scientific (2 mentions)
Advia chemistry xpt autoanalyzer, by Siemens (2 mentions)
Advia centaur autoanalyzer, by Siemens (2 mentions)
Cd27 pe cy7, by Thermo Fisher Scientific (2 mentions)
Luminex xmap analyser, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Lyosphere, by Qiagen (2 mentions)
Bicinchoninic acid bca assay, by Thermo Fisher Scientific (1 mentions)
Sandwich elisa kit, by R&D Systems (1 mentions)
Dextran sulfate sodium (dss), by MP Biomedicals (1 mentions)
Il 1β, by R&D Systems (1 mentions)
Gm csf, by R&D Systems (1 mentions)
Tnf alpha human uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
Il 6 human uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
Ifn gamma human uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
Il 1 beta human uncoated elisa kit, by Thermo Fisher Scientific (1 mentions)
19 protocols using procartaplex immunoassay kit
1
Cytokine Profiling by Luminex Assay
2
Cytokine Quantification by Luminex
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To determine cytokine concentrations, the cell culture supernatants were obtained from the culture plate wells and stored at – 80 °C. The concentrations of TNF-α, IL-12 and GM-CSF were determined using Luminex xMAP® with the Procartaplex Immunoassay kit (Affymetrix eBioscience) according to the manufacturer’s instructions.
3
Serum Biomarker Profiling in Bariatric Surgery
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Blood samples at baseline and 8 years after BS were collected after 12 h fasting. Serum was aliquoted and immediately stored at −80 °C. Serum glucose, cholesterol, triglycerides and HDL were analysed using an Advia Chemistry XPT autoanalyzer (Siemens Healthcare Diagnostics, Malvern, PA, USA). The LDL was calculated using the Friedewald equation. Serum insulin levels were measured via immunoassay using an ADVIA Centaur autoanalyzer (Siemens Healthcare Diagnostics, Malvern, PA, USA). Serum proteins, such as adhesion molecules (L-selectin, PECAM, ICAM-1, VCAM-1), metalloproteinases (MMP3, MMP9), growth factors (VEGF-A VEGF-D), adipokines (adiponectin, leptin), macrophage-related proteins (CD14, CD44, MBL, LVYE1) and other proteins (lactoferrin, elafin, NGAL, NRP1, PAI-1, RANTES) were analysed using ProcartaPlex immunoassay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) at baseline and 8 years after BS.
4
Multiplex Cytokine Profiling in Mice
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Cytokine levels in mouse sera were evaluated per manufacturer’s instructions using bead-based ProcartaPlex immunoassay kit (Thermo Fisher Scientific) consisting of either a custom-designed 10-plex mouse panel or a commercially available 36-plex mouse panel (Thermo Fisher Scientific). Analytes measured in the custom 10-plex included granulocyte CSF, IFNα, IFNγ, IL-12p70, IL-6, CXCL10, CCL2, CCL4, CCL7, and tumor necrosis factor–α.
5
PBMC Isolation, B Cell Purification, and Immunoassay
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For PBMCs and CD19+ B cells: PBMCs were isolated from 110 ml of whole blood by centrifugation over Ficoll. CD19+ B cells were isolated by positive selection using magnetic beads as previously described36 (link). Total RNA was extracted from each sample using an RNeasy mini kit (Qiagen) with quality assessed using an Agilent BioAnalyser 2100 and RNA quantification performed using a NanoDrop ND-1000 spectrophotometer.
For flow-sorted B cell samples from Espéli et al.37 (link): Flow sorting was performed using CD19-BV785, CD38-BV711, CD3-NC650, CD14-605NC, CD24-PerCP-Cy5.5, IgD-FITC. CD27-PE-Cy7 and Aqua (Invitrogen) (flow protocol outlined inExtended Data Figure 1 ), into sorting buffer (10mM Tris pH 8.0 and RiboLock RNase Inhibitor (1U/μL)) and frozen immediately.
Total IgA and IgE levels in patient serum were measured using a ProcartaPlex immunoassay kit (ThermoFisher) using 25ul of serum from each individual and run on a Luminex xMAP analyser. Raw data (MFI) were normalised to a concurrently measured 7 point standard curve according to the manufacturer’s instructions to return an absolute quantification (pg/ml). All measured values were encompassed by the standard distribution.
For flow-sorted B cell samples from Espéli et al.37 (link): Flow sorting was performed using CD19-BV785, CD38-BV711, CD3-NC650, CD14-605NC, CD24-PerCP-Cy5.5, IgD-FITC. CD27-PE-Cy7 and Aqua (Invitrogen) (flow protocol outlined in
Total IgA and IgE levels in patient serum were measured using a ProcartaPlex immunoassay kit (ThermoFisher) using 25ul of serum from each individual and run on a Luminex xMAP analyser. Raw data (MFI) were normalised to a concurrently measured 7 point standard curve according to the manufacturer’s instructions to return an absolute quantification (pg/ml). All measured values were encompassed by the standard distribution.
6
PBMC Isolation, B Cell Purification, and Immunoassay
7
Cytokine Profiling of Stimulated Blood
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Blood samples were taken to assess cytokine levels on day 0, week 2, week 6, and within 2 weeks of any progression. The 1 mL of whole blood was collected and stimulated with immune ligands (anti-CD3 as T-cells stimulant, and R848 as TLR 7/8 agonist to stimulate Natural Killer cells and foster T-cells’ response) on single lyophilized spheres (LyoSphere, Qiagen) within 8 hours from blood collection. Stimulated blood samples were incubated for 16 to 24 hours at 37°C and then centrifuged at 2000 to 3000 × g for 15 min to harvest the stimulated serum. Stimulated serum was stored at −20°C until the analysis and freeze-thaw cycles were minimized to preserve the quality of the samples. Serum levels of cytokines were measured either using the ProcartaPlex Immunoassay Kit (Luminex, ThermoFisher) or with custom-designed cartridges Ella (ProteinSimple). We measured 11 cytokines (IL-17A, IL-6, IL-1β, IFN-, IL-12p70, tumor necrosis factor-α (TNF-α), IL-10, IL-5, IL-4, IL-13 and GM-CSF) following the manufacturers’ instructions.22 (link)
8
Quantitative Analysis of Enteroendocrine Markers
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Protein supernatant extracted from 2D enteroids using RIPA buffer as well as basolateral media from enteroids cultured in 2D were analyzed for goblet cell marker Trefoil Factor 3 (TFF3) using a commercially available ELISA kit, per manufacturer’s instructions (R&D Systems, Minneapolis, MN). Additional proteins including Interleukin-4 (IL-4), Interleukin-8 (IL-8), Interleukin-18 (IL-18), soluble RANK ligand (sRANKL), ghrelin and Peptide YY (PYY) were analyzed using a custom ProcartaPlex Immunoassay Kit using antibody-based magnetic beads (ThermoFisher Scientific, Waltham MA). Samples were read and analyzed on a Luminex MAGPIX multiplexing system (Luminex, Northbrook, IL). Samples were normalized using protein expression as measured by a commercially available Bicinchoninic acid assay (BCA; ThermoFisher Scientific, Waltham MA).
9
Multiplex Cytokine Profiling in Mice
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Cytokine levels in mouse sera were evaluated per the manufacturer’s instructions using a bead-based ProcartaPlex immunoassay kit (Thermo Fisher Scientific) consisting of granulocyte CSF, IFN-α, IFN-γ, IL-12p70, IL-6, CXCL10, CCL2, CCL4, CCL7 and TNFα.
10
Serum Biomarker Profiling in Bariatric Surgery
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Blood samples at baseline and 8 years after BS were collected after 12 h fasting. Serum was aliquoted and immediately stored at −80 °C. Serum glucose, cholesterol, triglycerides and HDL were analysed using an Advia Chemistry XPT autoanalyzer (Siemens Healthcare Diagnostics, Malvern, PA, USA). The LDL was calculated using the Friedewald equation. Serum insulin levels were measured via immunoassay using an ADVIA Centaur autoanalyzer (Siemens Healthcare Diagnostics, Malvern, PA, USA). Serum proteins, such as adhesion molecules (L-selectin, PECAM, ICAM-1, VCAM-1), metalloproteinases (MMP3, MMP9), growth factors (VEGF-A VEGF-D), adipokines (adiponectin, leptin), macrophage-related proteins (CD14, CD44, MBL, LVYE1) and other proteins (lactoferrin, elafin, NGAL, NRP1, PAI-1, RANTES) were analysed using ProcartaPlex immunoassay kit (Thermo Fisher Scientific Inc., Rockford, IL, USA) at baseline and 8 years after BS.
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