Blood was centrifuged at 1500 × g for 10 min to isolate plasma. Multiplex analysis of cytokine production was determined using LEGENDplex (Biolegend), according to the manufacturer's instructions. Samples were acquired on a FACSVerse flow cytometer (BD Biosciences) and analyzed using the LEGENDplex v8 software. In a minority of samples that did not yield detectable cytokine levels, these samples were assigned the lowest limit of detection.
Legendplex
Manufactured by BD
Sourced in Germany
LEGENDplex is a multiplex assay platform that allows for the simultaneous quantification of multiple analytes in a single sample. The core function of LEGENDplex is to enable the detection and measurement of various biomolecules, such as cytokines, chemokines, and other protein targets, in a high-throughput and efficient manner.
Automatically generated - may contain errors
Lab products found in correlation
Legendplex, by BioLegend (4 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Facs lsrii flow cytometer, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Legendplex data analysis software, by BioLegend (1 mentions)
Fortessa x20, by BD (1 mentions)
Facscanto 2 cytometer, by BD (1 mentions)
Counting beads, by BioLegend (1 mentions)
Facscanto 2 cytofluorometer, by BD (1 mentions)
7 protocols using legendplex
1
Multiplex Cytokine Profiling in Plasma
2
Multiplex Assay for CXCL10 and CXCL11
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CXCL10 and CXCL11 in sera were assayed with a multiplex bead-based immunoassay LEGENDplex (Biolegend) according to manufacturer’s instructions. For data acquisition and analysis, a BD FACS LSR II flow cytometer and the LEGENDplex v7.0 data analysis software were used, respectively.
3
Cytokine Profiling in Primary Sclerosing Cholangitis
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IFN‐gamma, IL‐6, IL‐2, and IL‐4 were analyzed in serum samples from tubes with clotted venous blood. Sera from PSC‐patients and healthy controls were stored at −80°C until use. Cytokine concentrations were measured via a LEGENDplex™ bead‐based immunoassay using various anti‐human capture beads (IFN‐gamma, IL‐6, IL‐4, and IL‐2; Biolegend). Samples were analyzed on a FACSCanto II (BD Biosciences, Heidelberg, Germany) and cytokine levels were determined using the LEGENDplex™ cloud‐based software. Due to the limited availability of serum samples, we could not perform measurement of cytokines in every individual.
4
Cytokine and Chemokine Profile in Influenza Infection
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Concentration of cytokines and chemokines (IFN , IFN , TNF , IL-2, IL-6, IL-4, IL-5, IL-10, CCL2, CCL3, CCL4 and CCL5) were quantified on day 3 p.i.. BALF from mice infected with MA-HK68, P18-V2 and P18-N using a custom 13-plex LEGENDplex panel (Biolegend) according to the manufacturer's instructions. Samples were acquired by flow cytometry on BD LSRFortessa and analyzed using the LEGENDplex software.
5
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).
6
Multiplex Cytometric Analyte Profiling
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Analytes were measured by flow cytometry using Biolegend LegendPlex™ assays and a FACS Canto-II cytometer (Becton Dickson) and plotted using the ggplot2 (v3.3.5) [28 ], ggbreak (v0.0.8) [29 (link)], viridis (v0.6.2) [30 (link)], and rstatix (v0.7.0) packages. Details are provided in the Supplemental Methods.
7
Modulating CD19 CAR T Cell Cytokine Storm
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CD19 CAR T cells (1.5 × 103) were stimulated by anti‐CD3/CD28 mAb at for 24 hours. DEX and ruxolitinib were added initially at different concentrations (0, 1 and 10 µmol/L). CD19 CAR T cells were calculated using counting beads (424902, Biolegend, USA) by flow cytometry at 24 hours in different groups. CRS model was established by co‐culturing 5 × 104 Nalm6 cells, 1 × 104 monocytes and 5 × 104 CD19 CAR T cells as published in other group.28 The monocytes and CAR T cells were collected from the same donor. DEX and ruxolitinib were added at different concentrations (0, 1 and 10 µmol/L). T cells transduced with pCDH vector were used as a negative control. After 48 hours of coculture, cytokines were monitored in different dosages. The human cytokines included interleukin‐6 (IL‐6), IL‐10, IL‐2, IL‐4, IL‐7a, tumour necrosis factor‐α (TNF‐α), interferon‐γ (INF‐γ), granulysin, granzyme B, granzyme A, Perforin, sFas and sFasL were detected in the culture supernatants after co‐culture using the ‘LEGENDplex Human CD8 Panel’ bead‐based immunoassay (BioLegend, San Diego, USA) according to the manufacturer's protocol. Data were recorded by a FACSCanto II cytofluorometer (Becton Dickinson) and analysed using the ‘LEGENDplex’ Data Analysis software 8.0.
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