Cytokine concentrations were measured in cell culture supernatants using either FlowCytomix (eBioscience) or LegendPlex Mouse Th1/Th2 Panel (BioLegend) flow cytometry multianalyte detection system for IL-4, IL-2, IL-5, and IL-13 as per the manufacturer’s instructions.
Legendplex mouse th1 th2 panel
Manufactured by BioLegend
Sourced in United States
The LegendPlex Mouse Th1/Th2 Panel is a multiplex assay kit designed to measure the secreted levels of key Th1 and Th2 cytokines in mouse samples. The panel includes beads coated with antibodies specific to IL-2, IFN-gamma, TNF-alpha, IL-4, IL-5, and IL-13. This product allows for the simultaneous quantification of multiple analytes in a single sample, providing a comprehensive assessment of the Th1/Th2 immune response.
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Lab products found in correlation
Flowcytomix, by Thermo Fisher Scientific (3 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (1 mentions)
Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Sodium deoxycholate, by Merck Group (1 mentions)
Phosstop, by Roche (1 mentions)
Stainless steel beads, by Next Advance (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Granzyme b, by R&D Systems (1 mentions)
Bullet blender tissue homogenizer, by Next Advance (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Novocyte 2060, by Agilent Technologies (1 mentions)
Tissueruptor 2, by Qiagen (1 mentions)
Disposable probes, by Qiagen (1 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (1 mentions)
Streptavidin hrp, by BD (1 mentions)
Biotin rat anti mouse ige, by BD (1 mentions)
Rat anti mouse ige r35 72, by BD (1 mentions)
R35 118, by BD (1 mentions)
6 protocols using legendplex mouse th1 th2 panel
1
Cytokine Profiling of Cell Culture
2
Quantification of Immune Markers in Tumor Tissues
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4T1P and 4T1M tumor tissues were placed in a 1.6 mL tube containing RIPA buffer (5M NaCl (Fisher Chemical, Cat# 231-598-3), 0.5M EDTA (Sigma-Aldrich, Cat# EDS) pH=8, 1M Tris (Alfa Aesar, Cat# A12274) pH=8, 1% NP-40, 10% sodium deoxycholate (Sigma-Aldrich, Cat#D6750), 10% SDS (Fisher Scientific, Cat# BP2436-1), protease inhibitor cocktail (1:100, Roche, Cat# 11697498001) and phosphatase inhibitor (1:20 PhosSTOP, Roche, Cat# 4906845001). Stainless steel beads (Cat# SSB14B, Next Advance, New York, USA) were added and tumor tissue was homogenized using the Bullet Blender Tissue Homogenizer (Next Advance) according to the manufacturer’s protocol. The homogenate was centrifuged and supernatant was collected. The protein concentration of the tumor lysates was determined using Protein Assay Dye Reagent Concentrate (Bio-Rad, California, USA, Cat# 500-0006). The quantification of IFNγ and TNFα was carried out by using the LEGENDplex Mouse Th1/Th2 Panel (BioLegend, San Diego, CA, USA, Cat# 741053), in accordance with the manufacturer’s instructions. In addition, IFNα (Invitrogen, Cat# BMS6027) and Granzyme B (R&D Systems, Minneapolis, MN, USA, Cat# DY1865-05) were quantified by specific ELISA according to the manufacturer's instructions. All experiments were performed using at least three biological repeats.
3
Cytokine Profiling of Cell Culture
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Cytokine concentrations were measured in cell culture supernatants using either FlowCytomix (eBioscience) or LegendPlex Mouse Th1/Th2 Panel (BioLegend) flow cytometry multianalyte detection system for IL-4, IL-2, IL-5, and IL-13 as per the manufacturer’s instructions.
4
Protein Quantification from Frozen Brain
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Frozen brain tissue samples were homogenized in their tubes with a TissueRuptor II (6 speed, 30 s; Qiagen, USA) and disposable probes (Qiagen, USA) on ice with Halt Protease Inhibitor (ThermoFisher, USA) in N-PER reagent (ThermoFisher, USA). Homogenates were spun down thrice. The first spin in the 5 mL tubes was at 1000×g for 10 min at 4 °C. The supernatant was transferred into 2 mL tubes. The second and third spins were at 12,000×g for 15 min and 15,000×g for 20 min, respectively, both at 4 °C, with a transfer to new tubes after each spin. The protein samples were then processed with an 8-plex bead-based assay (LEGENDplex Mouse Th1/Th2 Panel; BioLegend, USA) according to manufacturer protocol. Samples were run on a Novocyte 2060 (ACEA Biosciences, USA). Quantification was normalized against a total protein bicinchoninic acid (BCA) assay (ThermoFisher, USA) of each protein sample.
5
Cytokine and Antibody Measurements
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Cytokine concentrations were measured in cell culture supernatants using either FlowCytomix (eBioscience) or a LegendPlex Mouse Th1/Th2 Panel (BioLegend) flow cytometry multi-analyte detection system for IL-4, IL-2, IL-5, and IL-13 per the manufacturer’s instructions. Serum IgE (and purified mouse IgE standard; BD) was captured overnight on a plate coated with 2 µg/ml rat anti–mouse IgE (R35-72; BD) and detected with biotin rat anti–mouse IgE at 1 µg/ml (R35-118; BD), streptavidin HRP (BD), and ABTS One Component HRP Microwell substrate (SurModics). H. polygyrus antigen (HEX) was obtained by homogenizing adult worms in PBS. Serum antigen-specific IgG1 was captured on a plate coated with 5 µg/ml HEX and detected using biotin rat anti–mouse IgG1 (Invitrogen), streptavidin HRP, and ABTS.
6
Quantification of Th1/Th2 Cytokines
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In serum samples, concentrations of Il10, interferon (Ifn)-g, Il4, and tumor necrosis factor (Tnf) were quantified either by specific enzyme-linked immunosorbent assay (R&D Systems, Abingdon, UK) or by the LEGENDplex Mouse Th1/Th2 Panel (BioLegend, San Diego, CA) according to the instructions of the manufacturers.
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