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Legendplex analysis software

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LEGENDplex analysis software is a data analysis tool designed to process and analyze data generated from BioLegend's LEGENDplex multi-analyte assay kits. The software provides users with the necessary functionality to visualize and interpret their experimental results.

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Lab products found in correlation

Legendplex bead array, by BioLegend (3 mentions) Legendplex multiplex cytokine panel kit, by BioLegend (2 mentions) Facscanto 2 flow cytometer, by BD (2 mentions) Cluster tubes, by Thermo Fisher Scientific (2 mentions) Legendplex immunoassay, by BioLegend (2 mentions) Accuri c6, by BD (2 mentions) Legendplex, by BioLegend (2 mentions) Legendplex mouse inflammation kit, by BioLegend (1 mentions) Cytoflex instrument, by Beckman Coulter (1 mentions) Facsaria, by BD (1 mentions) Neural tissue dissociation kit, by Miltenyi Biotec (1 mentions) Cellmatrix basement membrane gel, by American Type Culture Collection (1 mentions) Lsr 2, by BD (1 mentions) Gentlemacs dissociator, by Miltenyi Biotec (1 mentions) Mouse proinflammatory chemokine panel, by BioLegend (1 mentions) Tissuelyser, by Qiagen (1 mentions) Cell extraction buffer, by Thermo Fisher Scientific (1 mentions) Lsrfortessa flow cytometer, by BD (1 mentions) Protease inhibitor, by Merck Group (1 mentions) Facscanto 2, by BD (1 mentions)

13 protocols using legendplex analysis software

1

Multiplex Cytokine Analysis of Renal Cortical Extracts

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We used a LEGENDplexTM multiplex cytokine panel kit (BioLegend, San Diego, CA, USA) and a BD FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) to determine cytokine levels in the renal cortical protein extracts. These cytokines include interferon-γ (IFN-γ), interleukin (IL)-1A, IL-1B, IL-2, IL-6, and IL-17A. The fluorescent intensity of the target cytokine level was determined in each bead set by its fluorescent color code. Data generated by a flow cytometer was quantified by LEGENDplex analysis software (BioLegend). All samples were measured in duplicate.
Tain Y.L., Hou C.Y., Chang-Chien G.P., Lin S.F, & Hsu C.N. (2022). Perinatal Propionate Supplementation Protects Adult Male Offspring from Maternal Chronic Kidney Disease-Induced Hypertension. Nutrients, 14(16), 3435.
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2

Mouse Peripheral Blood Cytokine Measurement

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Mouse peripheral blood was extracted after right atrial puncture into a 2-ml Eppendorf tube. Then, blood tubes were incubated at room temperature for coagulation (15 min). After incubation, tubes undergo a centrifugation step (450g), and serum was collected. For cytokine measurement, we used LEGENDplex Mouse Inflammation Kit (BioLegend) following the manufacturer’s instruction. Data were acquired on a CytoFLEX instrument (Beckman Coulter) and analyzed using LEGENDplex analysis software (BioLegend).
Elyahu Y., Hekselman I., Eizenberg-Magar I., Berner O., Strominger I., Schiller M., Mittal K., Nemirovsky A., Eremenko E., Vital A., Simonovsky E., Chalifa-Caspi V., Friedman N., Yeger-Lotem E, & Monsonego A. (2019). Aging promotes reorganization of the CD4 T cell landscape toward extreme regulatory and effector phenotypes. Science Advances, 5(8), eaaw8330.
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3

Multiplex Cytokine/Chemokine Profiling in NHPs

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A 13-plex nonhuman primate specific cytokine/chemokine LEGENDplex bead array (BioLegend) was used according to the manufacturer protocol on plasma and BAL fluid samples. All samples were run on a BD FACS Aria and analysis was performed using LEGENDplex analysis software (Biolegend). An ELISA specific for cynomolgus/rhesus IFN-α2 was performed using BAL samples according to manufacturer protocol (PBL, VeriKine). BAL fluids from post-infection time points were diluted 50-fold in order to remain within the standard limits of detection. An anti-monkey albumin ELISA was used to quantify albumin concentrations in the BAL fluid and plasma (Innovative Research). Plasma was diluted according to the manufacturer protocol and BAL fluid was diluted either 1,000-fold for pre-infection samples or 20,000-fold for post-infection samples.
Wonderlich E.R., Swan Z.D., Bissel S.J., Hartman A.L., Carney J.P., O’Malley K.J., Obadan A.O., Santos J., Walker R., Sturgeon T.J., Frye LJ J.r., Maiello P., Scanga C.A., Bowling J.D., Bouwer A.L., Duangkhae P.A., Wiley C.A., Flynn J.L., Wang J., Cole K.S., Perez D.R., Reed D.S, & Barratt-Boyes S.M. (2017). Widespread virus replication in alveoli drives acute respiratory distress syndrome in aerosolized H5N1 influenza infection of macaques. Journal of immunology (Baltimore, Md. : 1950), 198(4), 1616-1626.
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4

Modulation of Microglia Activation

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Microglia were isolated from the brain from a naïve male rat (n=1) as previously described using the Neural Tissue Dissociation Kit and GentleMACS Dissociator (Miltenyi Biotec).10 Using Percoll centrifugation, myelin was removed from the cells. The brain cells were then cultured as previously described.10 First, we coated a 48-well plated with cell matrix basement membrane gel (ATCC, Manassas, VA). We then plated 2.5 ×105 cells/well in microglia-specific media (DMEM + 10% FBS), and media was changed every 48 hours. After 6 days, the cells were activated with LPS (1 μg/mL). 2 hours after activated, Treg (1:2 Treg:microglia), MSC (1:10 MSC:microglia) or combination of Treg (1:2) +MSC (1:10) were added to the wells; MSC were added either 1) concurrently with Treg or 2) 18 hours after activation.
The culture supernatant was collected at 72 hours. All samples were analyzed using a 13-plex rat-specific cytokine/chemokine LEGENDplex bead array according to the manufacturer’s protocol (Biolegend, San Diego, CA #740401). The samples were analyzed using a BD LSRII, and analysis was performed using LEGENDplex analysis software (Biolegend).
Caplan H.W., Prabhakara K.S., Toledano-Furman N.E., Zorofchian S., Kumar A., Martin C., Xue H., Olson S.D, & Cox CS J.r. (2020). Combination therapy with Treg and MSC enhances potency and attenuation of inflammation after traumatic brain injury compared to monotherapy. Stem cells (Dayton, Ohio), 39(3), 358-370.
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5

Quantifying Lung Chemokine Levels

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To determine the concentration of different chemokines in the lungs, mice were perfused with PBS, lungs were snap-frozen in liquid nitrogen and stored at −80 °C until further use. Lungs were homogenized in 1 ml of MEM supplemented with 5% FCS, penicilin, streptamycin, and glutamin and were lysed two times for 1.5 min at 25 Hz using a tissue lyser (Qiagen). Samples were centrifuged for 5 min at 3.5×g and supernatant was used. To determine the chemokine concentration the mouse pro-inflammatory chemokine panel (Biolegend) was used with v-bottom plate according to manufacturers protocol. Data was analzed with the Legendplex analysis software (Biolegend).
Welten S.P., Oderbolz J., Yilmaz V., Bidgood S.R., Gould V., Mercer J., Spörri R, & Oxenius A. (2021). Influenza- and MCMV-induced memory CD8 T cells control respiratory vaccinia virus infection despite residence in distinct anatomical niches. Mucosal Immunology, 14(3), 728-742.
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6

Multiplex Analysis of Plasma Cytokines

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Bead-based LEGENDplex immunoassay (BioLegend, London, UK) was used for the quantification of plasma cytokines and cytokines in cell culture supernatants. Procedures were performed according to the manufacturer’s protocol using cluster tubes (ThermoScientific, Loughborough UK). 12.5 μl of plasma from EDTA blood samples was used diluted 1:2 in assay buffer. Supernatants from bmMΦ were used non-diluted. Analyses were performed using a BD Accuri C6 (BD Biosciences, San José, USA) and LEGENDplex analysis software (BioLegend, San Diego, USA).
Moderzynski K., Heine L., Rauch J., Papp S., Kuehl S., Richardt U., Fleischer B, & Osterloh A. (2017). Cytotoxic effector functions of T cells are not required for protective immunity against fatal Rickettsia typhi infection in a murine model of infection: Role of TH1 and TH17 cytokines in protection and pathology. PLoS Neglected Tropical Diseases, 11(2), e0005404.
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7

Quantifying Cytokine Levels in Photostimulated Skin

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Skin from photostimulated Ai32 and TRPV1-Ai32 mice was harvested at 48 hours post stimulation. Tissue was homogenized in 500mL Cell extraction buffer (Invitrogen) supplemented with phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor (Sigma-Aldrich) according to the manufacturers recommendation. Tissue homogenates were centrifuged for 10 minutes at 7500 rpm. Cytokine levels in supernatant were quantified using a bead-based LEGENDplex immunoassay (Biolegend, San Diego, CA) according to the manufacturers protocol. Samples were analyzed on LSRFortessa flow cytometers (Becton Dickinson, Franklin Lakes, NJ). Data were analyzed using LEGENDplex analysis software (Biolegend, San Diego, CA).
Cohen J.A., Edwards T.N., Liu A.W., Hirai T., Jones M.R., Wu J., Li Y., Zhang S., Ho J., Davis B.M., Albers K.M, & Kaplan D.H. (2019). Cutaneous TRPV1+ Neurons Trigger Protective Innate Type-17 Anticipatory Immunity. Cell, 178(4), 919-932.e14.
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8

Multiplex Cytokine Profiling in Renal Cortex

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Cytokines IL-1A, IL-1B, IL-2, IL-6, IL-17A, and IFN-γ in the renal cortical protein extracts were measured using a LEGENDplexTM multiplex cytokine panel kit (BioLegend, SanDiego, CA, USA) [38 (link)]. Diluted samples were measured in duplicate using a BD FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). The fluorescent intensity of target cytokine concentration was identified in each bead set by its fluorescent color code. The data were quantified by LEGENDplex analysis software (BioLegend, San Diego, CA, USA).
Hsu C.N., Yu H.R., Chan J.Y., Lee W.C., Wu K.L., Hou C.Y., Chang-Chien G.P., Lin S, & Tain Y.L. (2022). Maternal Acetate Supplementation Reverses Blood Pressure Increase in Male Offspring Induced by Exposure to Minocycline during Pregnancy and Lactation. International Journal of Molecular Sciences, 23(14), 7924.
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9

Serum Cytokine Profiling by 13-Plex Mouse Inflammation Panel

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Serum from whole blood was collected, aliquoted and stored as described above. Following the manufacturer’s instructions, serum cytokines were assessed by 13-plex Mouse Inflammation Panel (740446, Biolegend, San Diego, CA, USA). All samples were analyzed in duplicate and technical replicates. Data were acquired on a 5-laster Beckman Coulter Cytoflex LX and analyzed using Biolegend’s Legendplex Analysis software (Version 1.1, San Diego, CA, USA).
Gopinath A., Mackie P.M., Phan L.T., Mirabel R., Smith A.R., Miller E., Franks S., Syed O., Riaz T., Law B.K., Urs N, & Khoshbouei H. (2023). Who Knew? Dopamine Transporter Activity Is Critical in Innate and Adaptive Immune Responses. Cells, 12(2), 269.
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10

Multiplex Cytokine Quantification in Kidney

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The cytokines interleukin IL-2, IL-6, IL-10, IL-17A, IL-17F, IL-22, and interferon-γ (IFN-γ) were measured in the kidney cortex homogenates using a LEGENDplex Custom 9plex cytokine panel kit (BioLegend, San Diego, CA, USA) according to the manufacturer’s instructions. The samples were analyzed on a BD FACS Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). Briefly, the tissue samples were diluted in the assay buffer and measured in duplicate. The color code of each bead identifies the target protein being assayed, while the fluorescent intensities on the beads measure target concentration. The data was quantified by LEGENDplex analysis software (BioLegend, San Diego, CA, USA). To adjust measurements for differences in total protein content of the samples, values are expressed in picograms per total gram of protein.
Hsu C.N., Hung C.H., Hou C.Y., Chang C.I, & Tain Y.L. (2021). Perinatal Resveratrol Therapy to Dioxin-Exposed Dams Prevents the Programming of Hypertension in Adult Rat Offspring. Antioxidants, 10(9), 1393.
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