The levels of 8 cytokine species (IL-2, IL-4, IL-6, IL-8, IL-10. INFγ, TNFα, GM-CSF) were measured in the serum of the 18 treatment-naïve CVID patients before and 24 h after the first Ig infusion, in the 24 maintenance therapy CVID patients and in 28 healthy controls using a commercially available kit (Bio-Plex Pro™ Human Cytokine 8-plex Assay, Hercules, CA, USA). The assay was performed according to the manufacturer's instructions and the concentrations of cytokines were calculated by comparing reads with a 5-parameter logistic standard curve using a Bioplex-200 instrument (Bio-Rad, Hercules, CA, USA).
Pro human cytokine 8 plex assay
Manufactured by Bio-Rad
Sourced in United States
The Pro Human Cytokine 8-plex assay is a multiplex immunoassay that allows for the simultaneous quantification of eight different human cytokines in a single sample. It is designed to provide a convenient and efficient method for analyzing multiple cytokines in research applications.
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Lab products found in correlation
Bio plex 200, by Bio-Rad (1 mentions)
Lipopolysaccharides (lps), by Thermo Fisher Scientific (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Permeabilization buffer, by BD (1 mentions)
Anti mouse cd8, by BioLegend (1 mentions)
Fixation permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
Manager 6, by Bio-Rad (1 mentions)
4 protocols using pro human cytokine 8 plex assay
1
Cytokine Profiling in CVID Patients
2
Cytokine Profiling of Engineered Tissue
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To identify biomarkers of inflammation, GM-CSF, IFN-γ, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-α were analyzed by measuring the secreted amount in the supernatant of both CTRL- and TENO-cultured cell-laden constructs after 3, 7 and 14 days of culture (N = 3). The conditioned medium of samples treated with 20 ng/mL LPS (Lipopolysaccharides; Invitrogen) for 24 h was used as positive control.
Cytokine release was tested through a multiplex enzyme-linked immunosorbent assay (ELISA) by using a Bio-Plex Pro Human Cytokine 8-plex assay. Each experimental sample was run in a biological duplicate. Cytokines were measured with a Bio-Plex200 System using the Bio-Plex ManagerTM software. All reagents and instruments including the Wash Station and Shaking Incubator were from BIORAD (Hercules, CA, USA).
Cytokine release was tested through a multiplex enzyme-linked immunosorbent assay (ELISA) by using a Bio-Plex Pro Human Cytokine 8-plex assay. Each experimental sample was run in a biological duplicate. Cytokines were measured with a Bio-Plex200 System using the Bio-Plex ManagerTM software. All reagents and instruments including the Wash Station and Shaking Incubator were from BIORAD (Hercules, CA, USA).
3
Multiparametric Flow Cytometry Analysis
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For the analysis of cell surface molecules, single-cell suspensions were prepared and fluorescently stained with the following antibodies from BioLegend: anti-human CD3 (317305), anti-human CD4(357146), anti-human CD8(344712), anti-human CD25(302609), anti-human CD56(362505), anti-mouse CD3(100203), anti-mouse CD4(100413), and anti-mouse CD8(100721). Tregs were first incubated with surface markers, washed, resuspended in 1 ml of cold Fixation/permeabilization buffer (eBioscience), and incubated at 4°C for 1 h. After washing with 2 ml of permeabilization buffer (eBioscience), cells were stained with Foxp3 antibodies at 4°C for 50 min protected from light. Finally, cells were washed with 2 ml of permeabilization buffer and detected on FACS CantoII (BD Biosciences). All flow cytometer data were analyzed by the FlowJo software.
The concentrations of IL1/2/4/6/8/10 and TNF-α in the serum of patients were measured using the Bio-Plex Pro Human Cytokine 8-plex assay according to the manufacturer’s instructions.
The concentrations of IL1/2/4/6/8/10 and TNF-α in the serum of patients were measured using the Bio-Plex Pro Human Cytokine 8-plex assay according to the manufacturer’s instructions.
4
Multiplex Cytokine Profiling for Biomedical Research
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Bio-Plex Pro™ Human Cytokine, 8-plex assay was used to measure the following cytokines; IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN gamma and TNF alpha. Briefly, 50ul of the magnetic beads were added to the pre-coated wells and washed twice. Samples and standards (50ul each) were added to the respective wells and incubated for two hours at room temperature with shaking at 850 rpm (rpm). Following the incubation, the plate was washed thrice and 25ul of detection antibody was added to each well. The plate was incubated for one hour at room temperature with shaking at 850 rpm. Fifty micro-liters of streptavidin-PE was added to each well following a wash and the plate was further incubated for 10 min at room temperature with shaking. After a final wash, 125ul of assay buffer were added to each well and the plate shaken for 30 s to re-suspend the beads. The plate was read using a MAGPIX™ and the fluorescence intensity (FI) from the immunoassay was acquired and data was analyzed using Bio-Plex manager 6.0. Concentrations that were lower than the low limit of detection were defined as non-measurable. The minimum detectable concentrations for each cytokine in this study were: IL-2; 10 pg/ml, IL-4; 1.5 pg/ml, IL-6; 1.9 pg/ml, IL-8; 2.9 pg/ml, IL-10; 3 pg/ml, GM-CSF; 60 pg/ml, IFN g; 1.1 pg/ml and TNF a; 4.8 pg/ml.
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