CII-specific T cells from draining LNs were enumerated and characterized using a DR1-CII tetramer [28 (link)]. Briefly, soluble DR1 covalently linked to the immunodominant CII peptide was produced in S2 cells, affinity-purified, biotinylated by BirA (Avidity, Aurora, CO, USA), and formed into multimeric units by stepwise addition of PE-labeled streptavidin (Rockland Immunochemicals, Limerick, PA, USA). For identification of CII-specific T cells ex vivo, LN cells were incubated with 1 μg of the tetramer at 37 °C for 2.5 h in complete HL-1 medium (50 U/ml penicillin G sodium, 50 μg/ml streptomycin sulfate, 0.05 mM β-mercaptoethanol, 2 mM l -glutamine, and 0.1 % bovine serum albumin; BioWhittaker/Lonza, Walkersville, MD, USA) supplemented with 5 mM NaN3. At the end of the incubation, antibodies specific for various CD markers were added and cells were incubated for an additional 30 minutes at 4 °C. Samples were then washed and resuspended in PBS supplemented with 0.1 % NaN3 and 2 % fetal bovine serum and analyzed by flow cytometry (BD LSR II). DR1-restricted, CII257–274-specific and HA306–318-specific T-cell hybridomas were used as positive and negative controls, respectively, to monitor the specificity and sensitivity of the DR1-CII tetramer (see Additional file 1 ).
β mercaptoethanol
Manufactured by Lonza
Sourced in United States, Switzerland
β-mercaptoethanol is a reducing agent used in various laboratory applications. It functions by breaking down disulfide bonds in proteins, which can be important for sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sodium pyruvate, by Merck Group (4 mentions)
L glutamine, by Lonza (3 mentions)
Penicillin streptomycin, by Lonza (3 mentions)
Hepes, by Lonza (2 mentions)
Gentamycin, by Lonza (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (2 mentions)
Rpmi 1640 medium, by PAN Biotech (2 mentions)
Non essential amino acids (neaa), by Lonza (2 mentions)
Lsr 2, by BD (1 mentions)
Bovine serum albumin (bsa), by Lonza (1 mentions)
Streptomycin sulfate, by Lonza (1 mentions)
Penicillin g sodium, by Lonza (1 mentions)
Escherichia coli o111 b4, by Merck Group (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
8 well chamber slide, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by BD (1 mentions)
Magnisort mouse cd4 t cell enrichment kit, by Thermo Fisher Scientific (1 mentions)
Anti il 4, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by BD (1 mentions)
8 protocols using β mercaptoethanol
1
Enumeration and Characterization of CII-Specific T Cells
2
Isolation and Stimulation of Murine T and B Cells
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T and B lymphocytes were isolated from the spleens of BALB/c mice (8-13 weeks old) using the Dynabeads untouched mouse T cell isolation kit (Thermo Fisher Scientific, Illkirch, France, 11413D) or the Miltenyi Pan B isolation kit II (Miltenyi Biotec, Bergisch-Gladbach, Germany, 130-090-862), respectively. 15-20×10 6 T cells and 20-35×10 6 B cells were isolated form the spleen of a mouse. The efficiency of isolation was determined by flow cytometry by specific B and T cell staining with a purity nearing 95%. The cells (2.5×10 6 ) were then stimulated with phorbol 12-myristate 13-acetate (abbreviated P, 50 ng mL -1 , Sigma, P8139) and ionomycin (abbreviated Iono or I, 1 µM, Sigma, I0634) or with purified NA/LE hamster anti-mouse CD3 (5 µg mL -1 , precoated, BD Biosciences, clone 145-2C11, 553057) and purified NA/LE hamster anti-mouse CD28 (5 µg mL -1 , BD Biosciences, clone 37.51, 553294) for T cells and lipopolysaccharide (LPS, 5 µg mL -1 , from Escherichia coli O111:B4, Sigma, L2630) for B cells. FLG was added at various concentrations either immediately or 2 h after the different activation parameters when indicated. Cells were incubated for 24 h in a 24 well plate at 37°C, 5% CO 2 in RPMI 1640 medium (Lonza BioWhittaker) supplemented with 10% FBS, 10 µg mL -1 gentamycin (Lonza BioWhittaker), 10 mM HEPES (Lonza BioWhittaker) and 0.05 mM β-mercaptoethanol (Lonza BioWhittaker).
3
Cytotoxicity Assessment of Nanomaterials
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HeLa (epithelial, human cervical adenocarcinoma) and Raw 264.7 (macrophages, Abelson murine leukaemia virus-induced tumour) cells were cultured as mono-layers in Dulbecco's modified Eagle medium supplemented with 10 µg/mL gentamycin (Lonza BioWhittaker), 10 mM HEPES (Lonza BioWhittaker), 0.05 mM β-mercaptoethanol (Lonza BioWhittaker) and 10 % FBS, in a humidified incubator (37 °C, 5% CO2). For toxicity experiments, cells were seeded in 24-well plates (approximately 0.5x10 5 cells/cm 2 , 1 mL/well). The nanomaterials were diluted in the cell culture media at different concentrations and subsequently cells were exposed for 24 h to the treatments. For confocal microscopy, the cells were seeded in 8-well chamber slide (Thermo Scientific) (approximately 1x10 4 cells/cm 2 , 500 µl/well). Cells were left to grow until 70-80 % confluency and exposed up to 24 h to the nanomaterials.
4
In vitro T cell differentiation
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Total and naive CD4+ T cells were isolated using the MagniSort Mouse CD4 T cell Enrichment Kit and MagniSort Mouse CD4 Naive T cell Enrichment Kit (Invitrogen), respectively. 5 × 105 cells in 200 µl RPMI-1640 medium (PAN Biotech) supplemented with 10% FCS (Biochrom), 2% sodium pyruvate (Sigma), 1× non-essential amino acids (Gibco), 0,01% β-mercaptoethanol (Roth), 1% penicillin/streptomycin (Lonza) and 2 mM L-glutamine (Lonza) were cultivated in 96-well U-bottom cell culture plates (Greiner) coated with 4 µg/ml anti-CD3 (BD Pharmingen) and supplemented with 1 µg/ml anti-CD28 (BD Pharmingen). For differentiation of the naive CD4+ T cells into Th1 lymphocytes, the culture was additionally supplemented with 10 ng/ml mIL-12 (Invitrogen) and 5 μg/ml anti-IL-4 (Invitrogen). Th2 cells were differentiated with 10 ng/ml IL-4, 5 µg/ml anti-IL-12, and 5 µg/ml anti-IFNγ. For Th17 differentiation 5 ng/ml TGFβ, 40 ng/ml IL-6, 10 ng/ml IL-23, 2 µg/ml anti-IFNγ, and 2 µg/ml anti-IL-4 were added to the medium, for regulatory T cell differentiation 5 ng/ml TGFβ, 20 ng/ml IL-2, 5 µg/ml anti-IL-12, 5 µg/ml anti-IFNγ, and 5 µg/ml anti-IL-4 were used. Iron sources ferric cloride FeCl3, ferric sulfate Fe2(SO4)3, and ferric citrate FeC6H5O7 were added at a concentration of 5 µM for 48 h.
5
Murine Bone Marrow Dendritic Cell Generation
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Bone marrow-derived dendritic cells (BMDCs) were differentiated in vitro from isolated bone marrow cells of uninfected 5-6-week old C57BL/6 mice. The cells were generated and cultured as described previously [17 (link)]. Briefly, using the BMDC differentiation method, bone marrow cells collected from mouse femurs and tibias were incubated for 7 days in RPMI media supplemented with 10% fetal bovine serum (Lonza), 100 unit/mL penicillin/streptomycin (Lonza), 0.1 mM nonessential amino acids (Lonza), 50 μM β-mercaptoethanol (Lonza), 1 mM sodium pyruvate (Sigma), 20 ng/mL GM-CSF (CreaGene, Gyeonggi, Republic of Korea), and 10 ng/mL IL-4 (CreaGene, Gyeonggi, Republic of Korea).
6
Murine Lymphoid Organ Preparation
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For all experiments, thymi and spleens were dissected and further processed under sterile conditions. Single-cell suspensions were obtained in RPMI 1640 with 10% (vol/vol) fetal calf serum (FCS), 2 mM l -glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 40 mM β-mercaptoethanol, 100 U ml−1 of penicillin, and 100 U ml−1 of streptomycin (all reagents from Lonza; solution hereafter referred to as complete medium).
7
Iron Supplementation Impacts T Cell Activation
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Spleens were isolated from tumor-naive female C57Bl/6N mice. After lysis of erythrocytes using the Mouse Erythrocyte Lysing Kit (R&D Systems) 2.5 × 105 splenocytes per well were then seeded in a 96-well round bottom plate and stimulated with 4 µg/ml plate-bound or 1 µg/ml soluble rat anti-mouse CD3 (clone 17A2; BD Pharmingen). Ferric chloride FeCl3 (Sigma Aldrich), ferric sulfate Fe2(SO4)3 (Sigma Aldrich), ferric citrate FeC6H5O7 (Sigma Aldrich), and holo-transferrin were added at concentrations of 2.5µM, 5 µM, 10 µM and 20 µM elementary iron. Splenocytes were cultured in RPMI-1640 medium (PAN Biotech) supplemented with 10% FCS (Biochrom), 2% sodium pyruvate (Sigma), 1× non-essential amino acids (Gibco), 0.01% β-mercaptoethanol (Roth), 1% penicillin/streptomycin (Lonza) and 2 mM L-glutamine (Lonza).
8
Generation of Bone Marrow-Derived Immune Cells
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Bone marrow-derived dendritic cells (BMDCs) and bone marrow-derived macrophages (BMDMs) were generated by flushing bone marrow cells from femurs and tibias. BMDCs were cultured for 6 days in Roswell Park Memorial Institute (RPMI) 1640 medium (Welgene Co., Daegu, Korea) supplemented with 10% fetal bovine serum (FBS) (Welgene), 100 unit/mL of penicillin/100 μg/mL of streptomycin (Welgene), 0.1 mM nonessential amino acids (Lonza, Basel, Switzerland), 50 μM β-mercaptoethanol (Lonza), 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO, USA), 20 ng/mL GM-CSF (CreaGene, Gyeonggi, Korea), and 10 ng/mL IL-4 (CreaGene). BMDMs were cultured for 6 days in Dulbecco’s modified eagle’s medium (Welgene) containing 10% FBS, 50 ng/mL mouse macrophage colony stimulating factor (M-CSF) (R&D System, Minneapolis, MN, USA), and 100 unit/mL of penicillin/100 μg/mL of streptomycin. Cultured cells were incubated at 37 °C in a 5% CO2 atmosphere.
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