Legendplex data analysis software

Manufactured by BioLegend

Sourced in United States, United Kingdom

LEGENDplex™ Data Analysis Software is a comprehensive data analysis tool designed to assist researchers in the interpretation of multiplex assay data. The software provides intuitive interfaces and analytical capabilities to support the efficient processing and visualization of complex immunological data.

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Lab products found in correlation

Legendplex, by BioLegend (15 mentions) Attune nxt, by Thermo Fisher Scientific (12 mentions) Facscanto 2 flow cytometer, by BD (7 mentions) Legendplex mouse inflammation panel, by BioLegend (6 mentions) Lsrfortessa, by BD (5 mentions) Cytoflex, by Beckman Coulter (4 mentions) Legendplex human anti virus response panel, by BioLegend (4 mentions) Attune nxt flow cytometer, by Thermo Fisher Scientific (4 mentions) Facsdiva, by BD (4 mentions) Legendplex multi analyte flow assay kit, by BioLegend (4 mentions) Facscanto 2, by BD (4 mentions) Canto 2 flow cytometer, by BD (4 mentions) Legendplex kit, by BioLegend (4 mentions) Facs canto 2, by BioLegend (3 mentions) Legendplex human inflammation panel 13 plex, by BioLegend (3 mentions) Facscalibur cytometer, by BD (3 mentions) Lsrfortessa flow cytometer, by BD (3 mentions) Human anti virus response panel 13 plex, by BioLegend (3 mentions) Legendplex bead based immunoassay, by BioLegend (3 mentions) Facsdiva software, by BD (3 mentions)

119 protocols using legendplex data analysis software

Cytokine Profiling in Synovial Fluid

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Synovial fluid mediators were determined by screening pro-inflammatory cytokines on three SF using a membrane-based Human Cytokine Array kit (ARY005B, R&D Systems, Minneapolis, MN, USA). The chemiluminescent signals were visualized with a ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA, USA) and analyzed using Image Lab 5.0. IL-6 and IFN-γ levels were quantified by ELISA according to the manufacturer's instructions (R&D Systems). Concentrations of MCP-1 levels were determined by Cytometric Bead Array (BD Biosciences, San Diego, CA, USA). Data was acquired on an LSRII cytometer (BD Biosciences) and analyzed using FCAP array v3 software (BD Biosciences). Quantification of IL-1β, IL-23, IL-12p70, IL-12p40, CCL17, CXCL10, IL-10, and IL-1RA in SF was performed by the LegendPlex Human M1/M2 Macrophage Panel (BioLegend, San Diego, CA, USA). Data was acquired on a MACSQUANT Q10 cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany) and analyzed using Legendplex Data Analysis Software (BioLegend).
IL-6 and TIMP3 were quantified in ADSC culture supernatants by ELISA (R&D Systems).

Sayegh S., El Atat O., Diallo K., Rauwel B., Degboé Y., Cavaignac E., Constantin A., Cantagrel A., Trak-Smayra V., Alaaeddine N, & Davignon J.L. (2019). Rheumatoid Synovial Fluids Regulate the Immunomodulatory Potential of Adipose-Derived Mesenchymal Stem Cells Through a TNF/NF-κB-Dependent Mechanism. Frontiers in Immunology, 10, 1482.

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IFNβ Quantification in Cell Culture

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Cell culture medium was collected and analyzed for IFNβ protein levels using multiplex mouse inflammation panel (LEGENDplex, Biolegend) as per manufacturer’s instructions. Data were collected on FACS Calibur two laser flow cytometer (Beckton Dickinson) and analyzed using LEGENDplex Data Analysis Software (Biolegend).

Xiao W., Klement J.D., Lu C., Ibrahim M.L, & Liu K. (2018). IFNAR1 controls autocrine type I interferon regulation of PD-L1 expression in myeloid-derived suppressor cells. Journal of immunology (Baltimore, Md. : 1950), 201(1), 264-277.

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Plasma Cytokine Profiling in ABA Mice

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Blood was collected from tail tip at 6 weeks of age, 5 days prior to the commencement of behavioral testing. Approximately, 350 μl of whole blood was collected into EDTA‐coated tubes (Microvette; Brand), which were centrifuged for 10‐min (8000 rpm, 4°C) within 30 min of collection and plasma separated and stored at −80°C until use. Following exposure to ABA conditions, and including 7 days of ad libitum food access and body weight recovery to >100% baseline to ensure that effects of susceptibility to ABA were not confounded by the acute effects of starvation, blood was collected via cardiac puncture. A custom rat multianalyte LEGENDplex bead‐based immunoassay kit was used to examine cytokine concentrations in plasma samples (LEGENDplex; BioLegend) that targeted six cytokines concurrently (IL‐6, IL‐10, IL‐4, IL‐1β, TNF‐α, RANTES). These analytes were selected based on their previously reported elevation in human AN patients and/or ABA mice. Plasma samples were screened with the LEGENDplex assay kit as per manufacturer's instructions, and the readout measurement acquired using a Fortessa X‐20 flow cytometer (Becton Dickinson [BD]). Data were analyzed using LEGENDplex Data Analysis Software (v8.0; BioLegend).

Milton L.K., Patton T., O'Keeffe M., Oldfield B.J, & Foldi C.J. (2022). In pursuit of biomarkers for predicting susceptibility to activity‐based anorexia in adolescent female rats. The International Journal of Eating Disorders, 55(5), 664-677.

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NK Cell Cytokine Secretion Assay

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Purified NK cells (2 × 10⁶ cells/mL) were cultured for 18 h with recombinant IL-12 (30 ng/mL) in combination with IL-15 (100 ng/mL) in the presence or absence of butyrate (1 mM). Supernatants were collected and assessed for cytokine content with a LEGENDplex human NK/CD8 cell panel (BioLegend, cat 740267) according to the manufacturer’s guidelines. Allowing quantification of TNFα, soluble Fas ligand (sFasL), IFNγ, granzyme A, granzyme B and perforin secreted by the NK cells. Samples were acquired using a FACS Canto II and data analyzed using LEGENDplex Data analysis software (BioLegend).

Zaiatz-Bittencourt V., Jones F., Tosetto M., Scaife C., Cagney G., Jones E., Doherty G.A, & Ryan E.J. (2023). Butyrate limits human natural killer cell effector function. Scientific Reports, 13, 2715.

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Multiplex Cytokine Measurement in Biological Samples

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We measured multi-cytokines by using a LEGENDplex multi-analyte flow assay kit (13-plex) for mouse inflammation panel (Biolegend). We assessed the cytokine concentrations in sera, liver homogenates, and supernatants of cultured cells according to the manufacturer’s instructions. We collected data using a BD FACSAria II and analyzed them with LEGENDplex data analysis software (Biolegend).

Xie D.L., Zheng M.M., Zheng Y., Gao H., Zhang J., Zhang T., Guo J.C., Yang X.F., Zhong X.P, & Lou Y.L. (2017). Vibrio vulnificus induces mTOR activation and inflammatory responses in macrophages. PLoS ONE, 12(7), e0181454.

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Cytokine and Antibody Profiling in Malaria

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Cytokines and chemokines (CXCL10 (IP-10), IFN-γ, IFN-β, IFN-α, IL-10, IL-12p40, IL-15, IL-18, IL-6, and TNF-α) were measured in the plasma by Legendplex according to manufacturer’s protocol (Biolegend, San Diego, CA). Plasma samples were collected at the indicated days and were stored in −80°C until analysis. All data were acquired on an LSR II Fortessa flow cytometer using FACSDiva software, version 8 (BD Biosciences, San Jose, CA). The concentration of each analyte is determined with cytometric data based on a standard curve using the Legendplex^™ data analysis software (Biolegend, San Diego, CA). P. chabaudi AJ-specific IgG was evaluated in plasma at indicated days by ELISA, as described previously [5 (link)]. Plates were coated with P. chabaudi AJ parasite lysate overnight at 4°C in PBS with 0.05% Sodium Azide. Plates were blocked with 1% BSA, 0.3% Tween 20, 0.05% Sodium Azide and then sera were incubated. Bound antibody was detected using Alkaline Phosphatase-conjugated goat anti-mouse IgG (Southern Biotech, Birmingham, AL). The reaction was revealed with a p-Nitrophenyl phosphate disodium salt (Sigma, USA) solution (1mg/ml) in diethanolamine buffer. Plates were analyzed with an Omega plate reader (FLUOstar Omega BMG LABTECH Inc, Durham, NC, USA).

Gbedande K., Ibitokou S.A., Ong M.L., Degli-Esposti M.A., Brown M.G, & Stephens R. (2023). Boosting Live Malaria Vaccine with Cytomegalovirus Vector Can Prolong Immunity through Innate and Adaptive Mechanisms. bioRxiv.

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Quantifying Th Cytokine Levels

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Plasma samples were evaluated for the abundance of interleukin 2 (IL2) and interferon gamma (IFNG, best known as IFNγ) with the LEGENDplex™ Human Th Cytokine Panel (BioLegend, #740001), as previously described [15 (link)]. Data were analyzed with the LEGENDplex™ Data Analysis Software (BioLegend).

Sugita M., Yamazaki T., Alhomoud M., Martinet J., Latouche J.B., Golden E., Boyer O., Van Besien K., Formenti S.C., Galluzzi L, & Guzman M.L. (2023). Radiation therapy improves CAR T cell activity in acute lymphoblastic leukemia. Cell Death & Disease, 14(5), 305.

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Cytokine Profiling of BMDM Responses

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Bone marrow was collected and BMDMs cultured as described before, but only for 48 h. Cells were then stimulated with 100 ng/ml LPS (Sigma-Aldrich) for 6 h. Cells were treated with mecamylamine, m-bromo PEP or vehicle (PBS or 0.1% DMSO, respectively) throughout the 48 h culture and 6 h stimulation. Supernatant was subsequently collected, and cytokine concentrations were assessed using LEGENDplex kits for TNFα, IL-1β, IL-6, IL-12 and IL-10 (BioLegend), as per the manufacturer’s instructions. Bead fluorescence was measured by flow cytometry using a CytoFLEX (Beckman Coulter, Pasadena, CA, USA), and analyzed using the LEGENDplex™ Data Analysis Software (BioLegend).

Godin J.R., Roy P., Quadri M., Bagdas D., Toma W., Narendrula-Kotha R., Kishta O.A., Damaj M.I., Horenstein N.A., Papke R.L, & Simard A.R. (2019). A silent agonist of α7 nicotinic acetylcholine receptors modulates inflammation ex vivo and attenuates EAE. Brain, behavior, and immunity, 87, 286-300.

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Cytokine Profiling of Macrophage Response

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MDM were incubated with 150 ng mL⁻¹ of PGA solution, 150 ng mL⁻¹ curdlan solution or 150 ng mL⁻¹ of curdlan nanoparticles for 4 hrs. Supernatants from MDM were assayed using a bead-based immunoassay that uses the principles of sandwich ELISA to quantify soluble analytes using a flow cytometer (LEGENDplex Multi-Analyte Flow Assay Kit, Human Inflammation Panel 1, BioLegend). The concentrations of IL-lβ, TNF-α, MCP-1, IL-8, IL-10, IL-12p70, Il-17, IL-18 and IL-23 were determined as per manufacturer’s instruction. Data were collected on a Fortessa five laser flow cytometer (Becton Dickinson) and analyzed using BioLegend’s cloud-based LEGENDplex™ Data Analysis Software.

Heo J., Sobiech T.A., Kutscher H.L., Chaves L., Sukumaran D.K., Karki S., Dube A., Prasad P.N, & Reynolds J.L. (2020). Hybrid Curdlan poly(γ-glutamic acid) nanoassembly for immune modulation in macrophage. Macromolecular bioscience, 21(1), e2000358.

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Plasma Cytokine and Endothelial Analysis

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Plasma samples were assayed for cytokine and endothelial marker analysis using LEGENDplex kits and the manufacturer’s protocols (Biolegend): “Human Anti-Virus Response Panel” (13-plex) at 1:2 dilution and “Human Vascular Inflammation Panel” (13-plex)”at 1:100 dilution in 5% TX100 (in PBS) for virus inactivation. Samples were run on a Novocyte Quanteon flow cytometer, and data analysis was conducted using BioLegend’s LEGENDplex Data Analysis Software.

Bergersen K.V., Pham K., Li J., Ulrich M.T., Merrill P., He Y., Alaama S., Qiu X., Harahap-Carrillo I.S., Ichii K., Frost S., Kaul M., Godzik A., Heinrich E.C, & Nair M.G. (2023). Health disparities in COVID-19: immune and vascular changes are linked to disease severity and persist in a high-risk population in Riverside County, California. BMC Public Health, 23, 1584.

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