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Elisa max deluxe set human ifn γ

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The ELISA MAX Deluxe Set Human IFN-γ is a laboratory equipment product designed for the quantitative measurement of human interferon-gamma (IFN-γ) levels in biological samples. It provides the necessary components to perform an enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of this specific cytokine.

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Lab products found in correlation

Human macsplex cytokine 12 kit, by Miltenyi Biotec (1 mentions) Facsaria fusion, by BD (1 mentions) Cytoflex s, by Beckman Coulter (1 mentions) Elisa max deluxe set human il 2, by BioLegend (1 mentions) Elisa max deluxe set human il 10, by BioLegend (1 mentions) Elisa max deluxe set human il 6, by BioLegend (1 mentions) Nod cg prkdcscid il2rgtm1wjl szj nsg mice, by Jackson ImmunoResearch (1 mentions) D luciferin, by Biosynth (1 mentions) Sunrise microplate reader, by Tecan (1 mentions) Typhoon 9410 laser flatbed scanner, by GE Healthcare (1 mentions) Prism 5, by GraphPad (1 mentions) Elisa max standard set human il 10, by BioLegend (1 mentions) Luciferase assay, by Promega (1 mentions) Ovcar3, by American Type Culture Collection (1 mentions) Jetoptimus system, by Polyplus Transfection (1 mentions) Jetprime system, by Polyplus Transfection (1 mentions) Image software, by Agilent Technologies (1 mentions) Gen5 microplate reader, by Agilent Technologies (1 mentions) Glomaxr instrument, by Promega (1 mentions) Infinite 200 pro, by Tecan (1 mentions)

14 protocols using elisa max deluxe set human ifn γ

1

Cytokine Profiling of Cell Supernatants

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Supernatants from cultured cells from the cross-presentation assay were monitored using the human MACS-Plex Cytokine 12 Kit purchased from Miltenyi Biotec (cat. #130-099-169). Acquisitions and analyses were performed on CytoFLEX S purchased from Beckman Coulter (cat. #B75442)/FACSAria Fusion purchased from BD Biosciences and FlowJo Software from Treestar, respectively. Supernatants from cultured cells from the peptide-based assay were monitored using ELISA tests purchased from BioLegend: ELISA MAX Deluxe Set Human IFNγ (cat. #430104), ELISA MAX Deluxe Set Human IL17 (cat. #433914), and ELISA MAX Deluxe Set Human IL9 (cat. #434705).
Fahrner J.E., Lahmar I., Goubet A.G., Haddad Y., Carrier A., Mazzenga M., Drubay D., Alves Costa Silva C., de Sousa E., Thelemaque C., Melenotte C., Dubuisson A., Geraud A., Ferrere G., Birebent R., Bigenwald C., Picard M., Cerbone L., Lérias J.R., Laparra A., Bernard-Tessier A., Kloeckner B., Gazzano M., Danlos F.X., Terrisse S., Pizzato E., Flament C., Ly P., Tartour E., Benhamouda N., Meziani L., Ahmed-Belkacem A., Miyara M., Gorochov G., Barlesi F., Trubert A., Ungar B., Estrada Y., Pradon C., Gallois E., Pommeret F., Colomba E., Lavaud P., Deloger M., Droin N., Deutsch E., Gachot B., Spano J.P., Merad M., Scotté F., Marabelle A., Griscelli F., Blay J.Y., Soria J.C., Merad M., André F., Villemonteix J., Chevalier M.F., Caillat-Zucman S., Fenollar F., Guttman-Yassky E., Launay O., Kroemer G., La Scola B., Maeurer M., Derosa L, & Zitvogel L. (2022). The Polarity and Specificity of Antiviral T Lymphocyte Responses Determine Susceptibility to SARS-CoV-2 Infection in Patients with Cancer and Healthy Individuals. Cancer Discovery, 12(4), 958-983.
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2

Cytokine Secretion Profiling of CAR-T Cells

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The E:T = 3:1 in vitro killing conditioned medium (as described in Section 2.4) was collected and divided into the T cell group, anti-CD19 CAR-T cell group, single KD anti-CD19 CAR-T cell group, double KD anti-CD19 CAR-T cell group, and tumor cell negative control group (1:1 mixture of K562 and K562-CD19 only). The secretion of IL-6, IFN-γ, IL-17, IL-2 and IL-10 was detected using ELISA MAX™ Deluxe Set Human IL-6 (Biolegend, Cat: 430504), ELISA MAX™ Deluxe Set Human IFN-γ (Biolegend, Cat: 430104), ELISA MAX™ Deluxe Set Human IL-17A kit (Biolegend, Cat: 433914), ELISA MAX™ Deluxe Set Human IL-2 (Biolegend, Cat: 431804), and ELISA MAX™ Deluxe Set Human IL-10 (Biolegend, Cat: 430604), respectively, following the manufacturer’s instructions.
Zhang H., Lv X., Kong Q, & Tan Y. (2022). IL-6/IFN-γ double knockdown CAR-T cells reduce the release of multiple cytokines from PBMCs in vitro. Human Vaccines & Immunotherapeutics, 18(1), 1-14.
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3

Evaluating CAR-T Cell Therapy in NSG Mouse Xenograft Model

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Twenty 6-8 weeks old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ NSG mice (The Jackson Laboratory #005557) were each given 0.5 × 106 JeKo-1_ffluc via i.v. (tail vein) injection on day 0. On day 5, the animals were i.p. injected with 150 mg/kg D-luciferin (Biosynth) and divided into 5 groups of 4 animals each by average bioluminescence. On day 6, each mouse was i.v. injected with 3 × 106 human PBMC (isolated from fresh buffy coats provided by OneBlood, Orlando, FL) and 5 × 106 CAR-Ts, and 6 h later, with i.p. injection of 1 μg switch 324N, which was increased gradually to 3 μg and 10 μg on days 7 and 8, respectively, and kept at 10 μg thereafter on days 10, 12, 14, 16, and 18. In cohort 5, 5 × 106 324-based conventional CAR-Ts were injected i.v. (tail vein) as positive control. To monitor IFNγ levels in mouse blood, 100 μL blood was collected by retro-orbital bleeding on day 9, 14, and 20 and diluted 5-fold for quantitative ELISA using the ELISA MAX Deluxe Set Human IFN-γ (BioLegend).
Peng H., Nerreter T., Mestermann K., Wachter J., Chang J., Hudecek M, & Rader C. (2022). ROR1-targeting switchable CAR-T cells for cancer therapy. Oncogene, 41(34), 4104-4114.
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4

Cytokine and Degranulation Assay

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Following a 48 h co-culture of cancer cells with HLA-matched PBMCs, the medium and PBMCs were harvested. IFN-γ and TNFα levels on harvested medium were analysed using ELISA Max Deluxe Set Human IFN-γ (BioLegend) and Human TNFα ELISA (Diaclone, Besancon Cedex, France) assays according to the manufacturers’ protocols. The absorbance at 450 nm was determined by the Sunrise microplate reader (TECAN, Männedorf, Switzerland). Degranulation assay on harvested PBMCs was performed utilizing CD107a staining by flow cytometry by analysing the percentages of stained cells. All experiments were performed in triplicate and repeated three times.
Polcaro G., Liguori L., Manzo V., Chianese A., Donadio G., Caputo A., Scognamiglio G., Dell’Annunziata F., Langella M., Corbi G., Ottaiano A., Cascella M., Perri F., De Marco M., Col J.D., Nassa G., Giurato G., Zeppa P., Filippelli A., Franci G., Piaz F.D., Conti V., Pepe S, & Sabbatino F. (2024). rs822336 binding to C/EBPβ and NFIC modulates induction of PD-L1 expression and predicts anti-PD-1/PD-L1 therapy in advanced NSCLC. Molecular Cancer, 23, 63.
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5

Evaluating Peptide Immunogenicity Using TILs

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To evaluate the immunogenicity of the identified peptides the identified peptides were synthesized, loaded on antigen presenting cells and co-cultured with the autologous TILs. All synthetic peptides were purchased from GeneScript as crude peptides. EBV-transformed B cells that express the correct HLA allele were loaded with the candidate peptides at a concentration of 10 μM for 2 h at 37 °C. Following three washing steps, the loaded B-cells were co-cultured with the autologous TILs in 1:1 ratio (105 cells) for an overnight incubation. The amounts of soluble IFNγ secreted from the TILs were measured by ELISA assay (ELISA MAX™ Deluxe Set Human IFNγ, Biolegend). Plates were scanned using the Typhoon-9410 laser flatbed scanner (GE Healthcare, USA) and analysed using MyAssays analysis software tool (www.myassays.com). Concentrations were calculated using four parameter logistic fit. From each peptide measurement we reduced the background measurement of the control, which was the same B-cells to which we added only DMSO and were later co-cultured with the TILs. All measurements were done in triplicates. A control peptide was used to normalize the concentration values between different ELISA plates of the same experiment. Graphs and statistics were done using GraphPad Prism 5.
Kalaora S., Lee J.S., Barnea E., Levy R., Greenberg P., Alon M., Yagel G., Bar Eli G., Oren R., Peri A., Patkar S., Bitton L., Rosenberg S.A., Lotem M., Levin Y., Admon A., Ruppin E, & Samuels Y. (2020). Immunoproteasome expression is associated with better prognosis and response to checkpoint therapies in melanoma. Nature Communications, 11, 896.
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6

Quantifying Secreted Cytokines by ELISA

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Secreted IL-10 or IFN-γ in the culture medium was measured via ELISA (ELISA MAX Standard Set Human IL-10, or ELISA MAX Deluxe Set Human IFN-γ, BioLegend, Koblenz, Germany), according to the manufacturer’s instructions.
Osanyinlusi S.A., Zischke J., Jacobs R., Weissinger E.M., Schulz T.F, & Kay-Fedorov P.C. (2022). Human Cytomegalovirus pUL11, a CD45 Ligand, Disrupts CD4 T Cell Control of Viral Spread in Epithelial Cells. mBio, 13(6), e02946-22.
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7

Cytotoxicity and IFNγ Assay for CAR T Cells

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We plated 105 target cells per well in a flat bottom 96 well plate in 100 μL culture media. The following day, we transfected the target cells with a luciferase expressing vector (Promega pGL4.51[luc2/CMV/Neo]) (RRID:Addgene_132962) using the JetPrime system (Polyplus Cat# 101000015) for tumor cells and the JetOptimus system (Polyplus Cat# 101000051) for primary healthy cells. The media was removed and replaced with fresh culture media 6-8 hours after transfection. The appropriate number of either OR2H1 CAR or mock transduced T cells were added the following day and co-cultured for 8 hours in the case of tumor cells and 24 hours for primary cultures (adipocytes, hepatocytes, and neurons). Cytotoxicity was measured via Luciferase Assay (Promega Cat# E1501). Cytotoxicity was calculated as (maximum viability control - individual well)/ (maximum viability control - maximum death control) *100 as a percentage.
In parallel, either H2009 (ATCC Cat# CRL-5911, RRID:CVCL_1514) or OVCAR3 (NCI-DTP Cat# OVCAR-3, RRID:CVCL_0465) cells were co-cultured with either OR2H1 CAR or mock transduced T cells at a ratio of 1:5. After co-culture of 8 hours, the supernatant was harvested and used for IFNγ detection using ELISA MAX™ Deluxe Set Human IFNγ (Biolegend 430101) and absorbance was measured at 450nm with Gen5 Microplate reader and Image Software (Biotek).
Martin A.L., Anadon C.M., Biswas S., Mine J.A., Handley K.F., Payne K.K., Mandal G., Chaurio R.A., Powers J.J., Sprenger K.B., Rigolizzo K.E., Innamarato P., Harro C., Mehta S., Perez B.A., Wenham R.M, & Conejo-Garcia J.R. (2022). Olfactory Receptor OR2H1 is an effective target for CAR T cells in human epithelial tumors. Molecular cancer therapeutics, 21(7), 1184-1194.
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8

Quantifying IFNγ Production by NK Cells

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For the detection of IFNγ, NK cell-containing cell-culture plates were centrifuged at 600 g rcf for 5 min at 4°C. 75 µl/well media was collected and stored at −20°C until analysis. ELISA MAX Deluxe Set Human IFN-γ (cat. 430115; BioLegend) was used according to the company’s protocol. An IFNγ standard curve was performed by serial twofold dilutions, starting from 10,000–4.88 pg/ml, while 0 pg/ml was used to calculate the background signal. 50 µl of the sample was used to detect IFNγ levels following the indicated stimulation.
Shemesh A., Pickering H., Roybal K.T, & Lanier L.L. (2022). Differential IL-12 signaling induces human natural killer cell activating receptor-mediated ligand-specific expansion. The Journal of Experimental Medicine, 219(8), e20212434.
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9

Glutor Modulates NK Cell Activation

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An amount of 0.1 × 106 resting or pre-activated NK cells were pre-treated with Glutor (100 nM) or DMSO (0.1%) for 30 min and afterwards stimulated via plate-bound CD16-mAb (2 µg/mL), NKp30 (2 µg/mL), NKG2D + 2B4 (2 µg/mL) or control IgG (2 µg/mL) or via IL-12 (0.25 ng/µL) + IL-18 (1.25 ng/µL) as previously described [13 (link)]. After 16 h supernatants were collected. IFN-γ secretion was analyzed using the ELISA MAX™ Deluxe Set Human IFN-γ by BioLegend according to the manufacturer’s instructions. Measurement was performed using a GloMaxR instrument (Promega, Fitchburg, WI, USA).
Picard L.K., Littwitz-Salomon E., Waldmann H, & Watzl C. (2022). Inhibition of Glucose Uptake Blocks Proliferation but Not Cytotoxic Activity of NK Cells. Cells, 11(21), 3489.
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10

CAR T Cell Cytokine Production Assay

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CAR T cells (16 h after mRNA electroporation) were co-cultured with target cells at an E:T ratio of 1:1 (25,000 cells each) in the absence or presence of 100 nM EGF or TGF-α at 37°C for 4 h. Next, the plates were centrifuged (450 g, 7 min, 4°C) and the supernatant stored at −80°C. IFN-γ was analyzed using the ELISA MAX Deluxe Set Human IFN-γ (BioLegend) according to the manufacturer’s instructions. Analysis was performed with an Infinite 200 PRO (Tecan).
Dobersberger M., Sumesgutner D., Zajc C.U., Salzer B., Laurent E., Emminger D., Sylvander E., Lehner E., Teufl M., Seigner J., Bobbili M.R., Kunert R., Lehner M, & Traxlmayr M.W. (2024). An engineering strategy to target activated EGFR with CAR T cells. Cell Reports Methods, 4(4), 100728.
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