Multiplex luminex-based immunoassay was performed for the cytokines IL-1β, IL-6, IL-8 and TNF-α using antibody-coated beads (Biosource International, Camarillo, CA, Luminex Corporation, Austin, TX) as indicators of SASP profile in amniotic fluid and maternal serum. Standard curves were developed with duplicate samples of known quantities of recombinant proteins that were provided by the manufacturer. Sample concentrations were determined by relating the absorbance that was obtained to the standard curve by linear regression analysis.
Antibody coated beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Antibody-coated beads are a type of laboratory equipment used for various applications. These beads have antibodies immobilized on their surface, which allows them to capture and isolate specific target molecules from a sample. The core function of these beads is to facilitate the separation and purification of target proteins, cells, or other biomolecules.
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Lab products found in correlation
Cd34 percp eflour710, by Thermo Fisher Scientific (1 mentions)
Thy 1 pe cy7, by BioLegend (1 mentions)
Fixable live dead nir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Novocyte quanteon, by Agilent Technologies (1 mentions)
Cd45 apc cy7, by BioLegend (1 mentions)
Anti cd3 fitc, by BioLegend (1 mentions)
4 protocols using antibody coated beads
1
Multiplex Cytokine Profiling in SASP
2
Multiplex Cytokine Quantification
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Multiplex luminex-based immunoassays were performed for the cytokines IL-6 and IL-8 with the use of antibody-coated beads (Biosource International, Camarillo, CA, Luminex Corporation, Austin, TX, USA). Standard curves were developed with duplicate samples of known quantities of recombinant proteins that were provided by the manufacturer. Sample concentrations were determined by relating the samples absorbances to the standard curve by linear regression analysis. Concentrations below the assay detection limits (IL-6 = 5.89 pg/mL and IL-8 = 5.93 pg/mL) were considered as half of each value.
3
Flow Cytometric Immunophenotyping of FLSs
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FLSs were blocked with 0.1 mg/mL mouse IgG (Jackson ImmunoReseach, West Grove, PA, USA) and 0.1 mg/mL rat IgG (Jackson ImmunoReseach) for 15 min and then surface stained with CD34-PerCP-eFlour710 (Clone: 4H11, Thermo Fisher Scientific, Waltham, MA, USA), CD45-APC-Cy7 (Clone: HI3, Biolegend), THY-1-PE-Cy7 (Clone: 5E10, Biolegend, San Diego, CA, USA), PDPN-PE (Clone: NZ-1.3, eBioscience, Thermo Fisher), and fixable LIVE/DEAD nIR Dead Cell Stain Kit (Life Technologies, Thermo Fisher Scientific). Data were acquired by NovoCyte Quanteon® (ACEA Bioscience Inc., San Diego, CA, USA) within 24 h of surface stain and processed in FlowJo (FlowJo software version 10.5.3). The spectral overlap was compensated using antibody-coated beads (eBioscience, San Diego, CA, USA). Gating was performed on live cells using fluorescence minus one (FMO).
4
Multiparameter Flow Cytometry of T Cell Activation
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CD4+ T cells were surface stained for 30 min using the following murine monoclonal antibodies: APC-anti 4-1BB (Cat. 309810), PE-anti Gal-3 (Cat. 126706), FITC-anti CD3 (BioLegend) and LIVE/DEAD (Life Technologies). Intracellularly staining were preceded by blocking with 50 µg/ml mouse IgG for 15 min before staining with an anti-TNFα antibody (Franklin Lakes, USA). Cells were washed, fixed and analyzed on a NovoCyte Quanteon™ or Amnis® ImageStream® Imagine Flow Cytometer. Spectral overlap was compensated using antibody-coated beads (eBioscience). Gating was done on live cells using fluorescence minus one (FMO). Data were analyzed using IDEAS for windows version 6.2 and/or FlowJo for Mac software version 10.1.
Imagestream gating included cells in focus, single cells and CD3 positive cells. A membrane mask was defined based on the CD3 stain using morphology mask and erode. Membrane mask included the outer three pixels. Within this mask the aggregation of 4-1BB was calculated and the surface specific MFI values determined.
Imagestream gating included cells in focus, single cells and CD3 positive cells. A membrane mask was defined based on the CD3 stain using morphology mask and erode. Membrane mask included the outer three pixels. Within this mask the aggregation of 4-1BB was calculated and the surface specific MFI values determined.
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