Concentrations of human cytokines GM-CSF, IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12p70, IL-17A, and tumor necrosis factor α in supernatants were analyzed with MACSPlex Cytokine Kits (Miltenyi Biotec) following the manufacturer’s instructions. The levels of these cytokines in the supernatants of M1 or M2 macrophage culture were measured 24 h after stimulation; the levels of IL12p70 in the supernatants of iMacs_IL-12 culture were measured 24 h after passaging; and the levels of IFN-γ in the supernatants of culture were measured 24 h after coculture.
Macsplex cytokine kit
Manufactured by Miltenyi Biotec
Sourced in Germany
The MACSPlex Cytokine kit is a multiplex assay designed to detect and quantify multiple cytokines simultaneously in a single sample. The kit uses magnetic bead-based technology to capture and measure cytokines, enabling efficient and high-throughput analysis of complex biological samples.
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Lab products found in correlation
Macsquantify 2, by Miltenyi Biotec (2 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (2 mentions)
Crp human elisa kit, by Thermo Fisher Scientific (1 mentions)
Facsdiva software, by BD (1 mentions)
Saponin, by Merck Group (1 mentions)
Foxp3 fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Brilliant stain buffer, by BD (1 mentions)
Quality control beads, by Beckman Coulter (1 mentions)
8 protocols using macsplex cytokine kit
1
Cytokine Profiling of Macrophage Subsets
2
Cytokine Production of BDCA2-CAR NK and T Cells
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Example 3
Cytokines Production of BDCA2-CAR NK and BDCA2-CAR T Cells
We analyzed the cytokines production of BDCA2-CAR-NK and BDCA2-CAR-T cells in the presence of target cells using MACSPlex cytokine kits from Miltenyi Biotec. BDCA2-CAR-NK cells produced very low or undetectable amounts of IFN-gamma and TNF-alpha after co-culturing with target cells without BDCA2 but produce higher amount of cytokines when co-culture with BDCA2 expressing target cells. (
3
Cytokine Profiling of PBMC and Neutrophil
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Supernatants from cultures of PBMC and neutrophil were tested for cytokine production by using the flow cytometry bead-based array with a MACSPlex Cytokine kit, according to the manufacturer’s instructions (Miltenyl Biotec, Bergisch Gladbach, Germany). MACSPlex Cytokine kit detects human IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, and TNF-α. Samples were developed using a MACSQuant Analyzer 10 and were evaluated using the Express Mode option of the MACSQuantify 2.8 software (Miltenyi Biotec, Bergisch Gladbach, Germany).
4
Cytokine and CRP Quantification
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Plasma were used for the quantification of IFN-γ, IL6 cytokines (MACSPlex Cytokine kit, Miltenyi, Bergisch Gladbach, Germany), and C-reactive protein (CRP Human ELISA kit, Thermofisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.
5
Tumor-Specific T-Cell Responses Evaluation
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The occurrence of spontaneous or vaccine-induced tumor-specific T-cells against survivin or mucin-1 peptides was assessed in both PBMCs and SKILs obtained from the DTH-skin test using methods described previously. First, in HLA*A02:01 positive patients, cells were stained with fluorescently labeled dextrameric complexes of MHC-I with relevant tumor peptides (Immudex, Virum, Denmark) (33 (link)). Two different fluorophores were used for staining and only the double-positive cells of the live CD8 T-cell population were considered antigen-specific to account for non-specific binding. Dextramers with peptides of survivin-1:5-14 (TLPPAWQPFL), survivin-1:95-105 (LTLGEFLKL) and Mucin (LLLLTVLTV) were used. Second, in all patients, functionality of SKILs was tested using a coculture assay with autologous PBMCs that were either pulsed with relevant peptide mixes (Survivin 1 PepTivator, Mucin-1 PepTivator) or unpulsed (16 (link)). In brief, cytokine production (IFN-gamma, IL-2, GM-CSF) by T-cells in response to coculture with PBMCs was measured using a cytometric bead array (MACSPlex Cytokine Kit, Miltenyi Biotec) and compared between pulsed and unpulsed PBMCs as a negative control. Based on the sensitivity of the assay and results from previous studies (33 (link)), a cytokine concentration of > 50 pg/ml compared to unpulsed PBMCs was considered biologically meaningful.
6
Cytokine Profiling of PBMCs and Neutrophils
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Serum levels of cytokines as well as the levels of cytokines secreted by mitogen-stimulated PBMCs and neutrophils were estimated by a flow cytometry bead-based array with a MACSPlex Cytokine kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The following cytokines were estimated: GM-CSF, IFN-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12, and TNF-α. Samples were processed in a MACSQuant Analyzer 10 and evaluated using the Express Mode option of the MACSQuantify 2.8 software (Miltenyi Biotec, Bergisch Gladbach, Germany).
7
Interleukin Assay of Secretome
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Total protein concentration was detected to normalize the secretome before interleukin assay. Interleukin assay was detected using MACSPlex Cytokine Kit (Miltenyi GmbH, Bergisch, Germany). Samples were prepared according to manufacture kit instructions and detected using BD FACS Diva Software.20 (link)
8
Multiparametric Immunophenotyping and Cytokine Analysis
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Post-culture cells were pelleted in V-bottom 96well plates and resuspended in 30 μl of antibodies at pre-optimized concentrations and diluted in Brilliant Stain Buffer (BD Biosciences), then incubated for 15' at RT. After a washing step, cell pellets were fixed either in FoxP3 fixation/permeabilization Buffer (ThermoFisher) for 20' at RT (AIM Assay) or in Formaldehyde 4% in PBS (ICS Assay). ICS was performed by incubating cells in 30 μl of antibodies against cytokines and Granzyme B diluted in a Saponin (Sigma-Aldrich) 0.5% W/V solution. The complete list of antibodies used for surface staining and ICS is shown in Table S2. Samples were acquired on a fully equipped Cytoflex LX within 6 hours from staining and fixation. Quality Control beads (Beckman Coulter) were used daily to check and standardize instrument performances. Data was analyzed with FlowJo v. 10.7
Cytokine measurements IL4, IL17, TNFa and IFNg were measured in frozen culture supernatants from the AIM assays described above by a bead-based sandwich ELISA (MACSPlex Cytokine Kit, Miltenyi Biotech) according to manufacturer instructions.
Cytokine measurements IL4, IL17, TNFa and IFNg were measured in frozen culture supernatants from the AIM assays described above by a bead-based sandwich ELISA (MACSPlex Cytokine Kit, Miltenyi Biotech) according to manufacturer instructions.
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