Naïve CD4+ T cells were isolated (EasySep Naïve CD4+ T cell isolation, StemCell) and incubated in mouse T cell medium (described in the “Silencing of eIF4E in OT2 T cells by lentivirus transduction” section) containing soluble anti-CD28 (1 μg/ml), cytokines, and anti-cytokine antibodies as detailed for polarization to TH1: mouse IL-12 (10 ng/ml), human IL-2 (50 U/ml), and anti-mouse IL-4 (10 μg/ml); TH2: mouse IL-4 (10 ng/ml), human IL-2 (50 U/ml), and anti-mouse IFN-γ (10 μg/ml); TH17: mouse IL-6 (20 ng/ml), mouse IL-23 (10 ng/ml), mouse IL-1β (10 ng/ml), human TGF-β1 (2 ng/ml), human IL-2 (50 U/ml), IL-4 (10 μg/ml), and anti-mouse IFN-γ (10 μg/ml); and Treg: mouse TGFβ (2 ng/ml) and human IL-2 (300 U/ml). TH1, TH2, and TH17 differentiation kits (Cytobox) along with human IL-2 were purchased from Miltenyi Biotec. mouse TGFβ was purchased from BioLegend. Naïve CD4+ T cells in conditions described above were activated on anti-CD3 (10 μg/ml)–coated plates for 3 days. Following differentiation, 20 μM 4EGI-1 was added to T cell cultures for 48 hours. Supernatant was removed and cytokine levels were determined using LEGENDPlex Mouse Th Cytokine Panel (BioLegend).
Legendplex mouse th cytokine panel
Manufactured by BioLegend
Sourced in United States
The LEGENDplex Mouse Th Cytokine Panel is a multiplex assay kit that allows for the simultaneous quantification of multiple mouse cytokines associated with T helper cell responses. The panel includes pre-mixed, pre-coated beads for the detection of key cytokines, such as IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17A, IFN-γ, and TNF-α.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria 3, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
Legendplex software v8, by BioLegend (2 mentions)
Concanavalin a (cona), by Merck Group (2 mentions)
Mouse tgfβ, by BioLegend (1 mentions)
Dimension vista 1500 system, by Siemens (1 mentions)
Accuri c6, by BD (1 mentions)
Lsr 2, by BD (1 mentions)
Th1 th2 th17 cytokine bead array, by BD (1 mentions)
Anti cd3 cd28 antibody, by Thermo Fisher Scientific (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Red blood cell lysing buffer, by Merck Group (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
L glutamine, by Lonza (1 mentions)
Nylon cell strainer, by BD (1 mentions)
Phorbol 12 myristat 13 acetate pma, by Merck Group (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Poly 1 c, by InvivoGen (1 mentions)
21 protocols using legendplex mouse th cytokine panel
1
T Cell Differentiation and Cytokine Profiling
2
Multiplex Cytokine Profiling in Mice
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Levels of IL-2, IL-4, IL-6, IL-9, IL-10, IL-22, TNF-α, and IFN-γ in mouse serum were determined using a LEGENDplex Mouse Th Cytokine Panel (BioLegend). Samples were assayed according to the BioLegend standard protocol and were examined using FACSAria III (BD Biosciences) driven by the FACSDiva software (BD Biosciences). The detection limitations of the cytokines were as follows: IL-6, IL-9, IL-10, and IFN-γ were > 0 pg/ml; TNF-α was > 3.26 pg/ml; IL-2 was > 2.99 pg/ml; IL-4 was > 1.52 pg/ml; and IL-22 was > 2.24 pg/ml.
3
Cytokine Quantification in Lung Samples
4
Murine Plasma Biomarker Profiling
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Whole blood samples were centrifuged for 10 minutes at 1500 × g and plasma was collected. Biochemical analysis was performed with an automated Dimension Vista 1500 System (Siemens Healthineers, Erlangen, Germany) and included aspartate aminotransferase, ALT, cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides. Plasma leptin and adiponectin concentrations were determined using the Mouse Leptin and Adiponectin Enzyme-Linked Immunosorbent Assay Kit (Thermo Fisher Scientific, Waltham, MA), respectively (N = 6 per group). Plasma cytokine concentrations were determined with the LEGENDplex Mouse Th Cytokine Panel (N = 6 per group; BioLegend, San Diego, CA). Analysis was performed on an Accuri C6 (BD Biosciences) and data were processed using the LEGENDplex Software v8.0 (BioLegend).
5
Cytokine Detection via CBA Assay
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For the detection of the cytokines, cytometric bead array (CBA) assay was performed using BioLegend LEGENDplex Mouse Th Cytokine Panel (BioLegend, catalog no. 741044) following the manufacturer's recommendations. Serum samples from experimental animals were diluted 1:4. BioLegend's LEGENDplex Mouse/Rat Free Active/Total TGF-β1 assay kit (catalog no. 740490) was used to measure total TGF-β1 in mouse serum. Following the manufacturer's recommendations, samples were treated to release free TGF-β1 from complex before continuing with the assay. Cell culture supernatants were not diluted. Data were acquired on a BD LSRII and analyzed using the LEGENDplex Data Analysis Software Suite.
6
Quantifying Cytokine Profiles of T Cells and Monocytes
7
Isolation and Cytokine Profiling of Murine Splenocytes
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Mononuclear cells were isolated from spleens by gentle extrusion of the tissue through a 50-μm-mesh Nylon cell strainer (BD). Cells were resuspended in DMEM medium that was supplemented with 10% fetal calf serum, 2 mM l -glutamine, 50 U/mg penicillin, and 50 U/mg streptomycin (Lonza, Levallois-Perret, France). Erythrocytes were lysed with red blood cell lysing buffer (Sigma-Aldrich).
For stimulation experiments, 2.5 × 106 cells per well were cultured for 48 h (37°C, 10% CO2) in DMEM medium in P24 plates that were precoated with anti-CD3/CD28 antibodies (4 µg/ml each; eBioscience). Culture supernatant was frozen at −80°C until processing.
Proteins from each colon were extracted with T-PER tissue protein extraction reagent (ThermoFisher Scientific) using a Fastprep instrument at 4,500 g for 30 s (two cycles). Samples were centrifuged at 500 g for 1 min, and supernatants were harvested for cytokine analysis. A cytometric bead array system (LEGENDplex Mouse Th Cytokine Panel, Biolegend) was used, according to manufacturer’s instructions, to determine the levels of the following cytokines: IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-γ, and TNF-α.
For stimulation experiments, 2.5 × 106 cells per well were cultured for 48 h (37°C, 10% CO2) in DMEM medium in P24 plates that were precoated with anti-CD3/CD28 antibodies (4 µg/ml each; eBioscience). Culture supernatant was frozen at −80°C until processing.
Proteins from each colon were extracted with T-PER tissue protein extraction reagent (ThermoFisher Scientific) using a Fastprep instrument at 4,500 g for 30 s (two cycles). Samples were centrifuged at 500 g for 1 min, and supernatants were harvested for cytokine analysis. A cytometric bead array system (LEGENDplex Mouse Th Cytokine Panel, Biolegend) was used, according to manufacturer’s instructions, to determine the levels of the following cytokines: IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFN-γ, and TNF-α.
8
Cytokine Production by Murine Immune Cells
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A total of 1 × 106 MC were cultured in 24-well plates and stimulated with either 100 ng/mL Pam2Cys (EMC microcollections, Tübingen, Germany), 100 ng/mL Pam3Cys (EMC microcollections, Tübingen, Germany), 10 ng/mL LPS from s.minnesota (Alexis, Lausen, Swiss), 0.5 µM CpG OD 1668 (Eurofins MWC Operon, Ebersberg, Germany), 10 ng/mL PolyIC (Invivogen, Toulouse, France), 10 ng/mL imiquimod (Invivogen), 10 ng/mL IL-33 (PeproTech, Hamburg, Germany) ionomycin (Sigma-Aldrich/Merck) or phorbol-12-myristat-13-acetate (PMA) (Sigma-Aldrich/Merck) for 24 h at 37 °C in MC-medium. Supernatants were quantified by enzyme-linked immunosorbent assay for IL-10 (R&D Systems, Minneapolis, MN, USA, detection limit 15.6–1000 pg/mL), IL-6 (R&D Systems, Minneapolis, MN, USA, detection limit 15.6–1000 pg/mL), TNF-a (R&D Systems, detection limit 10.9–700 pg/mL), LegendPlex Mouse Inflammation panel (BioLegend, San Diego, CA, USA) and LegendPlex Mouse Th Cytokine panel (Biolegend).
9
Quantifying Th Cytokine Release from Stimulated Splenocytes
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Cytokine release in the supernatant from stimulated splenocytes was quantified using the LEGENDplex Mouse Th Cytokine Panel (741,044-100T, Biolegend) according to the manufacturer’s guidelines using a V-bottom plate (Thermo Fisher Scientific). In short, 25 μL of vortexed capture beads were incubated with standards and samples on a shaker set at 800 rpm for 2 h at RT (protected from light) and spun down (250 × g, 5 min, RT). Beads were washed and incubated with 25 μL detection antibodies on a shaker set at 800 rpm for 1 h at RT (protected from light). An equal amount of streptavidin-phycoerythrin was added and incubated (shaker set at 800 rpm, 30 min, RT, protected from light), and beads were spun down (250 × g, 5 min), washed, and resuspended in 150 μL washing buffer before data acquisition using a Cytoflex flow cytometer (Beckman Coulter Life Sciences). The data were processed using Qognit (Qognit USA, accessed through Biolegend), a cloud-based software for analyzing multiplexed bead assays.
10
Mouse Serum Cytokine Profiling
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IL-2, IL-4, IL-17A, tumor necrosis factor (TNF)-α, IL-5, IL-22, and IFN-γ levels in the mouse serum were determined using a LEGENDplex Mouse Th Cytokine Panel (catalog no. 740005; BioLegend). All serum samples were diluted 1:1 in assay buffer. Samples were treated according to the BioLegend standard protocol and were examined using FACSAria III (BD Biosciences) driven by FACSDiva software (BD Biosciences). IL-12p70 was assayed by ELISA in microplates (catalog no. 1211202; DAKEWE BIOTECH CO., LTD.) according to the standard protocol.
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