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Legendplex human cd8 nk panel 13 plex

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The LEGENDplex™ Human CD8/NK Panel (13-plex) is a multiplex assay kit designed to simultaneously quantify the levels of 13 different human cytokines and chemokines in a single sample. The panel includes markers associated with CD8+ T cells and natural killer (NK) cells, providing a comprehensive analysis of the immune response.

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Lab products found in correlation

Legendplex data analysis software suite, by BioLegend (3 mentions) Legendplex software, by BioLegend (1 mentions) Cytoflex s, by Beckman Coulter (1 mentions) Easysep release human cd45 positive selection kit, by STEMCELL (1 mentions) Legendplex v8, by BioLegend (1 mentions) Elisa max kit, by BioLegend (1 mentions) Cytokine bead array, by BioLegend (1 mentions) Lsrfortessa x 20, by BD (1 mentions) Fix perm cell fixation cell permeabilization kit, by Thermo Fisher Scientific (1 mentions) Facsverse, by BD (1 mentions)

12 protocols using legendplex human cd8 nk panel 13 plex

1

Multiplex Cytokine Profiling of Tumor Cells

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Supernatants from tumor cell lysis experiments were collected after 16 h and stored at −20°C. A multiplex assay (LEGENDplex™ Human CD8/NK Panel (13-plex), BioLegend) was used according to the manufacturer's instructions. LEGENDplex™ Software from BioLegend was used for analysis of acquired data.
Jahnke S., Schmid H., Secker K.A., Einhaus J., Duerr-Stoerzer S., Keppeler H., Schober-Melms I., Baur R., Schumm M., Handgretinger R., Bethge W., Kanz L., Schneidawind C, & Schneidawind D. (2019). Invariant NKT Cells From Donor Lymphocyte Infusions (DLI-iNKTs) Promote ex vivo Lysis of Leukemic Blasts in a CD1d-Dependent Manner. Frontiers in Immunology, 10, 1542.
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2

Multiplex Cytokine Analysis in Ascites

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Concentrations of 13 cytokines and cytotoxic molecules were analyzed in ascitic supernatant (n = 53) using the LEGENDplex™ Human CD8/NK Panel (13-plex, BioLegend). The assay was performed according to the manufacturer’s instructions. Flow cytometric analysis was performed on CytoFLEX S (Beckman Coulter). Data were analyzed using online software (BioLegend).
Zhang J., He T., Yin Z., Shang C., Xue L, & Guo H. (2022). Ascitic Senescent T Cells Are Linked to Chemoresistance in Patients With Advanced High-Grade Serous Ovarian Cancer. Frontiers in Oncology, 12, 864021.
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3

Cytotoxic T Cell Stimulation Assay

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Tumour cells were seeded in complete RPMI medium one day prior to co-culture. Complete RPMI medium includes RPMI (Gibco 21870076), 10% fetal bovine serum, 1% l-glutamine, 1% penicillin-streptomycin. The next day, RPMI medium was replaced with T cell medium and antigen-specific T cells were seeded on top of the tumour cells at a 1:1 E:T ratio with IL-2 at 50 IU ml−1. Subsequent repeated co-cultures were set up every 48 h. For each co-culture, T cells were collected and counted using the Vi-CELL XR cell counter and viability analyser and replated onto fresh target tumour cells at a 1:1 E:T ratio. Before using the T cells for any assays, T cells were collected, counted and purified using EasySep Release Human CD45 positive selection kit (Stem Cell 100-0105) or purified by flow sorting. For ELISA experiments, after 5 stimulations of TCR T cells with target cells (A375), supernatant of co-cultures was collected and analyzed using the LEGENDplex Human CD8/NK Panel 13-plex (Biolegend 740267) according to the manufacturer’s instructions.
Carnevale J., Shifrut E., Kale N., Nyberg W.A., Blaeschke F., Chen Y.Y., Li Z., Bapat S.P., Diolaiti M.E., O’Leary P., Vedova S., Belk J., Daniel B., Roth T.L., Bachl S., Anido A.A., Prinzing B., Ibañez-Vega J., Lange S., Haydar D., Luetke-Eversloh M., Born-Bony M., Hegde B., Kogan S., Feuchtinger T., Okada H., Satpathy A.T., Shannon K., Gottschalk S., Eyquem J., Krenciute G., Ashworth A, & Marson A. (2022). RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature, 609(7925), 174-182.
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4

Quantifying Cell-Secreted Cytokines

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Secreted cytokines in the cell culture supernatant were quantified using a cytokine bead array suitable to detect IL-2, IL-4, IL-10, IL-6, IL-17A, TNF-α, sFas, sFasL, IFN-γ, Granzyme A, Granzyme B, Perforin and Granulysin (LEGENDplex® Human CD8/NK Panel 13-plex, BioLegend, San Diego, CA, USA). Experiments were performed according to the manufacturer’s instructions and as described previously [18 (link),19 (link)].
Lalia J.K., Schild R., Lütgehetmann M., Dunay G.A., Kallinich T., Kobbe R., Massoud M., Oh J., Pietzsch L., Schulze-Sturm U., Schuetz C., Sibbertsen F., Speth F., Thieme S., Witkowski M., Berner R., Muntau A.C., Gersting S.W., Toepfner N., Pagel J, & Paul K. (2023). Reduced Humoral and Cellular Immune Response to Primary COVID-19 mRNA Vaccination in Kidney Transplanted Children Aged 5–11 Years. Viruses, 15(7), 1553.
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5

Multiplex Cytokine Profiling in Cell Culture

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The supernatants from each of the wells of the cell culture experiments were collected and transferred into a 96-well plate using Biolegend LEGENDplex Human CD8/NK Panel (13-plex, Catalog#740267) according to the manufacturer’s instructions. Samples were run in BD LSRFortessa Flow cytometer and results were acquired using Legendplex v8.0 software (Biolegend, San Diego, CA, USA) [24 (link)].
Zhang Z., Yang A., Chaurasiya S., Park A.K., Lu J., Kim S.I., Warner S.G., Yuan Y.C., Liu Z., Han H., Von Hoff D., Fong Y, & Woo Y. (2021). CF33-hNIS-antiPDL1 Virus Primes Pancreatic Ductal Adenocarcinoma for Enhanced anti-PD-L1 Therapy. Cancer gene therapy, 29(6), 722-733.
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6

Profiling Cytokine Secretion in PBMC-eAPC Cocultures

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The supernatants of the PBMC‐eAPC cocultures were harvested at day 7, pooled from duplicate wells and stored at −20 °C. IL‐2, IL‐4, IL‐10, IL‐6, IL‐17A, TNF‐α, sFas, sFasL, IFN‐γ, granzyme A, granzyme B, perforin, and granulysin were determined using the LEGENDplex human CD8/NK panel (13‐plex, Biolegend) according to the manufacturer's instructions. The cytokine content of each donor is represented by a single dot.
Reithofer M., Rosskopf S., Leitner J., Battin C., Bohle B., Steinberger P, & Jahn‐Schmid B. (2020). 4‐1BB costimulation promotes bystander activation of human CD8 T cells. European Journal of Immunology, 51(3), 721-733.
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7

Cytokine Detection in Preclinical Models

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Human IFNγ (BioLegend, 430115), human IL2 (BioLegend, 431815), and mouse IFNγ (BioLegend, 430804) were detected utilizing the indicated BioLegend ElisaMax Kits, following the manufacturer's instructions. For human CAR T-cell coculture supernatants, Cytokine bead array (BioLegend, Legend Plex custom-made) was performed according to the manufacturer's protocol. For blood serum from NSG mice [see Establishment of human acute lymphoblastic leukemia xenograft mouse model], Cytokine bead array (BioLegend, LEGENDplex Human CD8/NK Panel, 13-plex, 741065) was performed according to the manufacturer's protocol. For blood serum from day 10 euthanized Balb-c mice (see Establishment of subcutaneous syngeneic colon carcinoma mouse model), Cytokine bead array (BioLegend, LEGENDplex MU Cytokine Release Syndrome Panel, 13-plex, 741024) was performed according to the manufacturer's protocol. The samples were detected using Fortessa LRS X20 (BD Biosciences) and analyzed using LEGENDplex Data Analysis Software Suite (BioLegend).
Righi M., Gannon I., Robson M., Srivastava S., Kokalaki E., Grothier T., Nannini F., Allen C., Bai Y.V., Sillibourne J., Cordoba S., Thomas S, & Pule M. (2023). Enhancing CAR T-cell Therapy Using Fab-Based Constitutively Heterodimeric Cytokine Receptors. Cancer Immunology Research, 11(9), 1203-1221.
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8

Cytokine Analysis of Cytotoxicity Assays

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Cytokine concentrations were analyzed from supernatants of cytotoxicity assays using the Human Th1/Th2/Th17 CBA Kit (human) Kit (BD, New Jersey, USA) and LEGENDplex™ Human CD8/NK Panel (13-plex) (Biolegend, San Diego, USA) following the manufacturer’s instructions.
Xiao X., Cheng Y., Zheng X., Fang Y., Zhang Y., Sun R., Tian Z, & Sun H. (2023). Bispecific NK-cell engager targeting BCMA elicits stronger antitumor effects and produces less proinflammatory cytokines than T-cell engager. Frontiers in Immunology, 14, 1113303.
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9

Multiplexed Cytokine Profiling in Co-Cultures

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Supernatants from 72 h co-culture were analyzed for multi-cytokine production utilizing LEGENDplex™ Human CD8/NK Panel (13-plex) (741065, Biolegend) according to manufacturer’s protocol. The samples were detected using LSRFortessa X20 (BD Biosciences) and analyzed using LEGENDplex™ Data Analysis Software Suite (Biolegend).
Ferrari M., Righi M., Baldan V., Wawrzyniecka P., Bulek A., Kinna A., Ma B., Bughda R., Akbar Z., Srivastava S., Gannon I., Robson M., Sillibourne J., Jha R., El-Kholy M., Amin O.M., Kokalaki E., Banani M.A., Hussain R., Day W., Lim W.C., Ghongane P., Hopkins J.R., Jungherz D., Herling M., Welin M., Surade S., Dyson M., McCafferty J., Logan D., Cordoba S., Thomas S., Sewell A., Maciocia P., Onuoha S, & Pule M. (2024). Structure-guided engineering of immunotherapies targeting TRBC1 and TRBC2 in T cell malignancies. Nature Communications, 15, 1583.
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10

Multiplex Cytokine Quantification in Cell Culture

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Detection of cytokines in the cell culture supernatant of stimulated cells was performed using LEGENDplex™ Human CD8/NK Panel (13-plex, BioLegend) suitable for detection of IL-2, IL-4, IL-10, IL-6, IL-17A, TNF-α, sFas, sFasL, IFN-γ, Granzyme A, Granzyme B, Perforin, Granulysin, according to the manufacturer’s instructions. Briefly, freshly prepared provided cytokine standard or thawed cell culture supernatant was mixed with cytokine-specific beads, incubated for 2 hours and washed. After sequential incubation of bead-bound cytokines with biotin-labeled cytokine detection antibodies and streptavidin PE antibodies, non-binding antibodies were washed off and PE-labeled bead-bound cytokines were subsequently analyzed by flow cytometry. Quantification of cytokines was carried out using the standard. The data was analyzed using the online LEGENDplex™ Data Analysis Software of the manufacturer. The assay was performed in duplicates and mean values of each sample were used for further analysis.
Paul K., Sibbertsen F., Weiskopf D., Lütgehetmann M., Barroso M., Danecka M.K., Glau L., Hecher L., Hermann K., Kohl A., Oh J., Schulze zur Wiesch J., Sette A., Tolosa E., Vettorazzi E., Woidy M., Zapf A., Zazara D.E., Mir T.S., Muntau A.C., Gersting S.W, & Dunay G.A. (2022). Specific CD4+ T Cell Responses to Ancestral SARS-CoV-2 in Children Increase With Age and Show Cross-Reactivity to Beta Variant. Frontiers in Immunology, 13, 867577.
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