Twelve-well flexiPERM culture masks (Greiner Bio-one) were fixed to the plasma polymerised slides and incubated with streptavidin (Sigma; 5 μg/ml, 4°C, overnight). Wells were then washed (seven times, 1 × PBS), blocked (0.01% BSA, 30 mins) and washed again (seven times, 1 × PBS). Saturating levels of biotinylated ligands were incubated on streptavidin-coated surfaces for 90 mins at room temperature, and washed seven times with 1 × PBS. The biotinylated ligands included anti-CD3 (5 μg/ml, clone 145-2C11; eBioscience), anti-CD28 (2.5 μg/ml, clone 37.51; eBioscience) and anti-CD3(Fab) (1/50 dilution, clone 145-2C11; as prepared above).
Clone 145 2c11
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Clone 145-2C11 is a laboratory equipment product. It is used for specific applications related to cell biology and immunology research. The core function of this product is to enable researchers to perform relevant experiments and analyses. A detailed description of the product's features and specifications is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Clone 37, by Thermo Fisher Scientific (19 mentions)
Anti cd3, by Thermo Fisher Scientific (8 mentions)
Anti cd28, by Thermo Fisher Scientific (7 mentions)
Anti cd28 antibody, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640 medium, by Lonza (3 mentions)
Streptomycin, by Merck Group (3 mentions)
Penicillin, by Merck Group (3 mentions)
U bottom culture plates, by Greiner (3 mentions)
Il 23, by R&D Systems (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Recombinant murine il 4, by R&D Systems (3 mentions)
Clone xmg1, by Thermo Fisher Scientific (3 mentions)
Clone 11b11, by Thermo Fisher Scientific (3 mentions)
Monensin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (3 mentions)
Recombinant murine il 2, by R&D Systems (2 mentions)
Neutralizing anti il 4, by Thermo Fisher Scientific (2 mentions)
Anti cd3e, by Thermo Fisher Scientific (2 mentions)
Cd4 cd62l t cell isolation kit 2, by Miltenyi Biotec (2 mentions)
42 protocols using clone 145 2c11
1
Immobilized Ligand Binding Assay
2
T Cell Proliferation Assay
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CD4+ T cells were purified from lungs and spleen of BeO-exposed and spleen of PBS-treated mice at day 21. Tconv cells were purified from naive spleen. Teffs (CD44hiCD25lo) and Tregs (CD25+) were sorted from purified CD4+ T cells on FACS ARIA II fusion (BD Biosciences). Tconv cells were stained with 1 μM carboxy fluorescein isothiocyanate (CFSE; Invitrogen) and cocultured with Teffs or Tregs in a 1:1 ratio in a 96-well anti-CD3–coated (1–2 μg/mL, clone 145-2C11, Thermo Fisher Scientific) plates for 5 days. Transwell assays were performed in a 96-well transwell plate (Corning). Loss of CFSE was examined by flow cytometry at day 5.
3
Isolation and Priming of Murine CD8+ T Cells
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Spleens from male or female OT-I/Cas9 mice were harvested and mechanically dissociated using a 100 μm and 70 μm cell strainer (Corning). The cell suspension was washed by centrifugation at 1000 xg using an isolation buffer (0.1% BSA in PBS). Red blood cells were lysed using a red blood cell lysis buffer (155 mM NH4Cl, 10 mM NaHCO3, 0.1 mM EDTA in distilled water; all Sigma) for five minutes. Cells were then washed once in PBS and once in isolation buffer and resuspended in isolation buffer. CD8 T cell isolation was performed using the Dynabeads Untouched Mouse CD8 Cells kit (11417D, Invitrogen) according to manufacturer’s instructions. Isolated naïve CD8 T cells were then resuspended in mouse CD8 T cell medium (RPMI, 10% FBS, 100 U/ml of Penicillin-Streptomycin, 10 ng/ml IL-2 (12340026, ImmunoTools), 0.5 ng/ml IL-7 (12340075, ImmunoTools), 1 ng/ml IL-15 (12340155, ImmunoTools) and 50 μM 2-mercaptoethanol (Merck)) at a concentration of 1x106 cells/ml and primed using plate-bound CD3 antibody (0.25 μg/ 2x106 cells, clone 145-2C11, Thermo Fisher Scientific) and CD28 antibody (2.5 μg/ 2x106 cells, clone 37.51, Thermo Fisher Scientific) for 48 h. T cells were then either retrovirally transduced (see below) or maintained daily at a density of 1x106 cells/ml for approximately 10 days before performing experiments (T cell stimulation by CD3 antibody or tumor-antigen).
4
T-cell Exhaustion Marker Analysis
5
Cytokine Secretion Profiling of Splenocytes
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Upon single cell suspension of splenocytes and normalizing counts, cells were plated on a 12-well flat bottom plate at 107 cells/mL and incubated for 72 h at 37°C with cRPMI containing stimulation. Stimulation conditions include unstimulated media only control (used to subtract background signal), Endofit OVA (10 μg/mL), or positive control anti-CD3e (0.5 μg/mL, Invitrogen, clone 145-2C11) and anti-CD28 (0.25 μg/mL Invitrogen, clone 37.51). After incubation, supernatant was collected and frozen for analysis of cytokine secretion by ELISA. Th1/Th2 mouse uncoated ELISA kits from Invitrogen were used and kit instructions were followed to analyze secreted IL-4, IFNγ and IL-10. Samples were run as technical duplicates. For analysis of cytokine production, 1 pg/mL was added to all data points to assess on log scale.
6
Isolation and Sorting of Immune Cell Subsets
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Cells of the nose and NT were further processed to isolate CD45+ cells by magnetic separation using a CD45 MACS kit (Miltenyi Biotec). The efficacy of the MACs sort was 95% or higher. For cell sorting experiments purified CD45+ cells were rested for 30 mins in a 37°C, 5% CO2 incubator before surface staining with antibodies specific to CD3 (Invitrogen; clone 145-2C11), CD19 (Invitrogen; clone 1D3), CD11b (Invitrogen; clone M1/70), F480 (Invitrogen; clone BM8), Ly6G (Invitrogen; clone 1A8) and sorted into CD3+, CD19+, CD11b+ F480+, CD11b+ Ly6G+ cell populations using a BD FACsAria Fusion cell sorter. Cells were washed in sterile PBS and RNA was extracted from cells as described.
7
Cytokine Production by T Cells in Response to Allergen Stimulation
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8 × 105 cells derived from spleen were cultured (200 μl/well) in U-bottom culture plates (Greiner, Frickenhausen, Germany) using RPMI 1640 medium (Lonza, Verviers, Belgium) with 10% FCS, penicillin (100 U/ml)/streptomycin (100 μg/ml) (Sigma). All cells were stimulated with culture medium as a negative control, a polyclonal stimulation with anti-CD3/CD28 (1 μg/ml, clone 145-2C11 and clone 37.51, eBioscience) or allergen-specific stimulation with PE (100 μg/ml). Interleukin (IL)-5, IL-10, IL-13 and Interferon-γ (IFN-γ) production by T cells were determined after 48 h (anti-CD3/CD28) or 96 h (PE) incubation. Culture supernatants were collected and stored at − 20 °C until further analysis with the Ready-SET-Go!® ELISA (eBioscience) according to the manufacturer’s instructions.
8
Murine CD4+ T cell differentiation
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Single cell suspensions were prepared from the lymph node and spleen of C57BL/6 mice and CD4+ T cells were isolated using the CD4+ T Cell Isolation Kit (130-104-454, Miltenyi Biotec). Cells were cultured in RPMI medium supplemented with 10% FBS, 50 μM 2-mercaptoethanol and 10,000 U/ml penicillin/streptomycin. Cells were activated with plate-bound anti-mouse CD3 (5 μg/ml; clone 145-2C11), and soluble anti-mouse CD28 (2 μg/ml; clone 37.51, eBioscience) in RPMI for four days in the following differentiation media:-
(a) for Th1 - 10 ng/ml IL-12 (200–12), 10 ng/ml IL-2, 5 µg/ml anti-IL-4 (504122, BioLegend);
(b) for Th17 - 50 ng/ml IL-6 (216–16), 10 ng/ml IL-1β (211-11B), 10 ng/ml IL-23 (200–23), 5 ng/ml TGF-β (100–21), 5 µg/ml anti-IL-4, 5 µg/ml anti-IFN-γ (505834, BioLegend);
(c) for Tregs - 10 ng/mL IL-2 (200-02, PeproTech) and 10 ng/mL TGF-β DMSO or the indicated concentration of PQS was added from day 1 of stimulation and cell populations were analysed by FACS after 4 days
(a) for Th1 - 10 ng/ml IL-12 (200–12), 10 ng/ml IL-2, 5 µg/ml anti-IL-4 (504122, BioLegend);
(b) for Th17 - 50 ng/ml IL-6 (216–16), 10 ng/ml IL-1β (211-11B), 10 ng/ml IL-23 (200–23), 5 ng/ml TGF-β (100–21), 5 µg/ml anti-IL-4, 5 µg/ml anti-IFN-γ (505834, BioLegend);
(c) for Tregs - 10 ng/mL IL-2 (200-02, PeproTech) and 10 ng/mL TGF-β DMSO or the indicated concentration of PQS was added from day 1 of stimulation and cell populations were analysed by FACS after 4 days
9
Th1 and Th17 cell differentiation
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CD4+ T cells from C57BL/6 mice were cultured in Iscove’s modified Dulbecco’s medium with KnockOut Serum Replacement (both from Invitrogen) for 5 days in the presence of TH1 [IL-12 (12 ng/ml) and anti–IL-4 (10 μg/ml)] or TH17 [TGF-β (3 ng/ml), IL-6 (30 ng/ml), IL-1β (10 ng/ml), IL-23 (10 ng/ml), anti–IL-4 (10 μg/ml), and anti–IFN-γ (10 μg/ml)] conditions and anti-mouse CD3 (1.0 μg/ml; clone 145-2C11, eBioscience) and anti-mouse CD28 (0.5 μg/ml; clone 37.51, eBioscience). Mouse BALB/cAnNcrl CD4+ T cells were cultured in TH2 [IL-4 (40 ng/ml) and anti–IFN-γ (10 μg/ml)] conditions in RPMI 1640. Neutralizing antibodies were supplied from BioLegend. All cytokines were supplied by PeproTech, except IL-23 (R&D Systems). All mice were obtained from Harlan UK. Secreted cytokines were measured using the MSD platform according to the manufacturer’s instructions.
10
Isolation and Activation of Murine Immune Cells
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The WT, PTENflox, PTENM−KO, β-cateninflox, and β-cateninM−KO mice were anesthetized with sodium pentobarbital (100 mg/kg, i.p.), and then Bio-Gel elicited peritoneal macrophages were isolated as described previously (22 (link)). The macrophages were cultured in medium (Invitrogen) supplemented with 10% FBS, 100 μg/ml of penicillin/streptomycin (Life Technologies; Grand Island, NY). The peripheral blood or spleen T cells were purified using the EasySep™ mouse T cell isolation kit (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions. T cells were then stimulated with anti-CD3 (1 μg/ml, Clone 145-2C11) and anti-CD28 (2 μg/ml, Clone 37.51) (eBioscience).
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