Blood samples (cohort #3) were collected in plasma separation tubes (BD Diagnostics), and plasma was collected by centrifugation, stored at 5 °C for 5 days, aliquoted, and stored long term at −80 °C. Plasma concentrations of tPA, interleukin (IL)-6, IL-8, P-selectin, D-dimer, PSGL-1, and sCD40L were measured by LEGENDplex bead-based immunoassay (BioLegend, product #740891) [60 (link),61 ], according to the manufacturer’s instructions. Plasma levels of MRP8/14, MPO, VCAM1, and cystatin C were measured by a LEGENDplex bead-based immunoassay (BioLegend, product no. 740551), according to the manufacturer’s instructions.
Legendplex bead based immunoassay
Manufactured by BioLegend
Sourced in United States
LEGENDplex is a bead-based immunoassay for the simultaneous detection and quantification of multiple analytes in a single sample. The product utilizes color-coded beads and fluorescent-labeled detection antibodies to enable the measurement of multiple targets in a small sample volume.
Automatically generated - may contain errors
Lab products found in correlation
Legendplex data analysis software, by BioLegend (3 mentions)
Duoset sandwich elisa kit, by R&D Systems (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Quantstudio 3 real time pcr system, by Thermo Fisher Scientific (2 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (2 mentions)
Human cd8 nk panel, by BioLegend (2 mentions)
Negative magnetic bead selection, by STEMCELL (2 mentions)
L ap4, by Merck Group (2 mentions)
Canto 2 flow cytometer, by BD (2 mentions)
Immunodeficient nsg mice, by Jackson ImmunoResearch (1 mentions)
Facs influx, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Pst tubes, by BD (1 mentions)
Plasma separation tubes, by BD (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Rmil 33, by R&D Systems (1 mentions)
Rmil 25, by R&D Systems (1 mentions)
Rmil 2, by BioLegend (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
38 protocols using legendplex bead based immunoassay
1
Biomarker analysis of plasma samples
2
Evaluating CAR T Cell Efficacy in Tumor-Bearing Mice
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Immunodeficient NSG mice (Jackson Laboratory, ME) were implanted subcutaneously with MDA-MB-231 cells and injected intravenously with CAR T cells and then FITC-folate (as indicated) when tumors reached ~100 mm3 in size. Mice were maintained on folic acid-deficient diet (TD.95247, Envigo) in order to reduce the level of folic acid in mice to a physiological levels found in humans45 (link),46 (link). Tumors were measured every other day with calipers, and tumor volume was calculated according to equation: Tumor volume = ½(L × W2) where L is the longest axis of the tumor and W is the axis perpendicular to L. Mouse blood was also collected to measure cytokine levels (e.g., IL-2, IL-6, IFNγ, and TNFα) using LEGENDplex bead-based immunoassay (Biolegend, CA) and systemic toxicity was monitored by measuring bodyweight loss. All animal care and use followed by National Institutes of Health (NIH) guidelines and all experimental protocols were approved by the Purdue Animal Care and Use Committee.
3
Enrichment and Activation of NK Cells
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NK cells were enriched using magnetic cell isolation methods (Miltenyi Biotec, Bergisch Gladbach, North Rhine‐Westphalia, Germany) that depleted CD19+ and CD3+ cells from the splenocyte pool. NK cells (CD19−CD3−NK1.1+) were then isolated to over 95% purity by FACS on an FACS Influx or an FACS Aria (BD Biosciences, Franklin Lakes, NJ, USA) cell sorter. Purified NK cells were resuspended to a concentration of 106 cells mL−1 and cultured with or without recombinant human IL‐12 (1 ng mL−1; mouse cross‐reactive) and mouse IL‐18 (5 ng mL−1). Supernatants were collected after 6 h. IFNγ concentrations were measured using the LEGENDplex bead‐based immunoassay (BioLegend, San Diego, CA, USA) performed according to the manufacturer's instructions, with the exception that we used half the recommended volumes.
4
Biomarker Profiling of Whole Blood
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The remainder of the whole blood samples were centrifuged, plasma removed, and then frozen at -80 °C until subsequent batch analysis of the systemic level of the markers of VD. Concentrations of the myoglobin (Myo), myeloid-related protein 8/14 (MRP8/14), lipocalin A (NGAL), matrix metalloproteinases (MMP) 2 and 9, osteopontin (OPN), myeloperoxidase (MPO), serum amyloid A (SAA), insulin-like growth factor-binding protein (IGFBP) 4, intercellular adhesion molecule (ICAM) 1, vascular cell adhesion molecule (VCAM) 1, and cystatin C (Cys) were measured by flow cytometry using LEGENDplex bead-based immunoassay (BioLegend, Ssan Diego CA, USA). Acquired raw data were analyzed using LEGENDplex Data Analysis Software v.7 (VigeneTech, Carlisle, MA, USA).
5
Plasma MRP8/14 Quantification Protocol
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Blood samples were collected in PST tubes (BD Diagnostics), and plasma was collected by centrifugation, stored at 5°C for 5 days, aliquoted, and stored long term at −80°C. Plasma levels of MRP8/14 were measured by a LEGENDplex bead-based immunoassay (BioLegend, product no. 740891), according to the manufacturer’s instructions.
6
Multiplex Cytokine Analysis in Nasal Samples
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Inflammatory cytokines IL-1β, IFN-α2, IFN-γ, TNF-α, MCP-1, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-18, IL-23, and IL-33 were measured in the nasal wash samples collected from patients and controls by BioLegend's LEGENDplex™ bead-based immunoassay, according to the manufacturer's instructions (BioLegend). In brief, samples and standards were thawed at room temperature and then incubated with mixed capture beads in a filter plate on a shaking platform for 2 h at room temperature. Following incubation, the plate was washed and sequentially incubated with detection antibodies and SA-PE for 1 h and 30 min, respectively. After final washes, the beads were resuspended in wash buffer and transferred into FACS tubes. Samples were then read on a BD LSRFortessa flow cytometer (BD Biosciences). Data was analyzed using the LEGENDplex Data Analysis Software according to the manufacturer's instructions (BioLegend).
7
Measuring TNF Concentrations
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TNF concentrations were analyzed using the LEGENDPLEX bead-based immunoassay (Biolegend) according to the manufacturer’s instructions.
8
Cytokine Profiling of CII-Treated Cells
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The supernatants were collected after 96 h of cell culture with CII. The cytokine levels were analyzed using the LEGENDplex bead-based immunoassay (BioLegend, CA, USA), according to the protocol supplied by the manufacturer. In brief, 20 μl of assay buffer, standard or sample, and mixed capture beads was added to the wells of a 96-well plate and the plate was shaken at 800 rpm for 2 h. The beads were washed with 200 μl of wash buffer. 20 μl of detection antibodies was added to the wells and the plate was shaken at 800 rpm for 1 h. Then, 20 μl of PE-conjugated streptavidin was added to the wells and the plate was shaken at 800 rpm for 30 min. After washing the beads with wash buffer, the standard and samples were read on a FACSVerse flow cytometer, and the data were analyzed with the software provided by BioLegend.
9
Culturing and Activating Kidney ILC2s
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FACS-purified kidney ILC2s (defined as CD45+Lin−CD127+KLRG1+) from IL-33–treated mice were cultured (∼10,000 cells/well) in a 96-well U-bottom plate in Iscove’s Modified Dulbecco’s Medium containing 5% FCS, 1% penicillin/streptomycin, and 50 µM 2-mercaptoethanol (all Gibco) in the presence of rmIL-2 (10 ng/ml, BioLegend) with or without all trans RA (1 µM; Enzo Life Sciences). In some experiments, the plate was precoated with the Notch ligand DLL4 (2.5 µg/ml; R&D) for at least 3 h at 37°C. Cells were cultured for 4 d and analyzed for surface marker expression using flow cytometry. In some experiments, cells were stimulated with either rmIL-25 (1 ng/ml; R&D) or rmIL-33 (1 ng/ml) on day 3 and culture supernatants were collected 24 h later and analyzed for IL-5 and IL-13 protein with the LEGENDplex bead-based immunoassay according to the manufacturer’s instructions (BioLegend).
10
Cytokine Profiling in Viral Infection
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