T2 cells (ATCC CRL-1992), were stained with CTV (following the method explained in CD8+ T cell proliferation assay above) and subsequently pulsed with 1 μg/mL of pWT1126. T2 cells were co-cultured overnight with untransduced or transduced CB-CD8+ T cells in a 1:1 or a 1:10 ratio (T:E cells). After 14−16 h, cells were stained with 7-AAD (BD Pharmingen), after which, flow cytometry analysis was performed on a BD LSRFortessa using FACSDiva, with acquisition of a fixed amount of total volume for each sample. Cytokines in supernatants were measured using the LEGENDplex assay (BioLegend). Flow cytometry analysis was performed on a BD LSRFortessa using FACSDiva, and post-acquisition analysis was performed with LEGENDplex Data Analysis Software.
Legendplex data analysis software
Manufactured by BD
Sourced in United States
LEGENDplex Data Analysis Software is a tool that enables users to analyze data generated from LEGENDplex assays. It provides functionalities for data processing, visualization, and management.
Automatically generated - may contain errors
Lab products found in correlation
Legendplex, by BioLegend (2 mentions)
Legendplex assay, by BioLegend (1 mentions)
7 aad, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Legendplex multiplex assay, by BioLegend (1 mentions)
Cellquest software, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
4 protocols using legendplex data analysis software
1
T cell Cytotoxicity and Cytokine Assay
2
Cytokine Profiling of BEAS-2B and HSAEC Secretomes
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BEAS-2B and HSAEC supernatants were screened for inflammatory cytokines associated with SASP (IL-1β, MCP-1, IL-6, IL-8) using bead-based immunoassays LEGENDplex™ (740809, Biolegend), following the manufacturer’s protocol. Briefly, cell supernatants were incubated for 2 h with two sets of specific anti-cytokine antibodies-conjugated beads, differentiated by size and internal fluorescence intensities, followed by a 1 h incubation with a detection antibody. After 30 min incubation of the mix with streptavidin-conjugated phycoerythrin, the fluorescent signal intensity, which is proportional to the amount of bound analytes, was detected using a FACSVerse™ flow cytometer (BD Biosciences) and data were analyzed with the LEGENDplex Data Analysis Software.
3
Multiplex Analysis of Secreted Proteins
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Secreted interleukin-6 (IL-6), dickkopf-related protein 1 (DKK-1), and osteoprotegerin (OPG) were analyzed in the supernatant of each sample with a customized human BioLegend’s LEGENDplex™ multiplex assay (Biolegend, San Diego, CA, United States) containing antibodies for IL-6, DKK-1, and OPG. Analysis was done following the manufacturer’s instructions, and internal standards served to determine the concentration of each protein. The multiplex assay was measured with a BD FACSVerse™ (Becton, Dickinson and Company, Franklin Lakes, NJ, United States) and analyzed with the LEGENDplex™ Data Analysis Software. The measured protein concentration was normalized to each supernatant’s total protein content (see 2.7.1 ).
4
Quantification of Basal Cytokine Release
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Basal release of IL‐1β, IFN‐α2, IFN‐γ, TNF‐α, MCP‐1 (CCL2), IL‐6, IL‐8 (CXCL8), IL‐10, IL‐12p70, IL‐17A, IL‐18, IL‐23 and IL‐33 was quantified in the conditional medium after 4‐h and 24‐h culture of 1 × 106 K562 cells/mL, respectively, 5 × 105 K562 cells/mL, using the Human Inflammation Panel (LEGENDplex™, BioLegend) bead‐based immunoassay according to manufacturer's instructions. Samples were analysed with a BD FACSCalibur™ using the BD CellQuest™ software (Becton Dickinson) and LEGENDplex™ Data Analysis Software. Cytokine release quantification was adjusted to the cell number.
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