Murine or human total or CD4 T cells were isolated by magnetic bead negative and cultured in RPMI 1640 media supplemented with 10% FBS, 2 mM glutamine, 10 mM HEPES and 55 μM β-mercaptoethanol. T cells were stimulated on plates with 5 μg/mL anti-CD3ε and anti-CD28 antibodies (Pharmingen) for 24 or 48 hrs as indicated. Naïve T cells were cultured with 10 ng/mL IL-7. Activated T cells were washed off of stimulation plates and cultured with 5 ng/mL IL-2 for assays.
Anti cd3ε and anti cd28 antibodies
Manufactured by BD
Anti-CD3ε and anti-CD28 antibodies are laboratory reagents used in cell culture and research applications. These antibodies bind to specific cell surface proteins, CD3ε and CD28, which are important for T cell activation and function. The core function of these antibodies is to provide a controlled and reproducible method for stimulating T cells in vitro.
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Lab products found in correlation
Cell strainer, by BD (1 mentions)
Mouse cd4 t cell isolation kit, by STEMCELL (1 mentions)
Xanthine oxidase, by Merck Group (1 mentions)
Rhil 2, by Thermo Fisher Scientific (1 mentions)
Biotin anti mouse cd4, by Thermo Fisher Scientific (1 mentions)
Live dead fluorochrome conjugated antibodies, by Thermo Fisher Scientific (1 mentions)
Tnf α duoset elisa kits, by R&D Systems (1 mentions)
β actin, by Merck Group (1 mentions)
Penicillin streptomycin, by BD (1 mentions)
70 μm nylon mesh strainer, by BD (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti cd3ε and anti cd28 antibodies
1
T Cell Activation and Expansion Protocol
2
Isolation and Activation of Murine CD4+ T Cells
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Female C57BL/6 mice were purchased from the Model Animal Research Center of Nanjing University. All animal procedures conformed to the principles outlined in the Guide for the Care and Use of Medical Laboratory Animals [50 ]. Spleens were meticulously processed over a cell strainer (Falcon) and washed in PBS supplemented with 2% fetal bovine serum (FBS, Gibco) to achieve a homogeneous single-cell suspension. Total CD4+ T cells were then isolated from the splenic cell suspension using the Mouse CD4+ T Cell Isolation Kit (Stemcell Technologies), as per the manufacturer's instructions. The isolated cells were cultured in a medium enriched with 10% FBS and 1% penicillin/streptomycin (HyClone), and stimulated with plate-bound anti-CD3ε and anti-CD28 antibodies (5 μg/ml, BD Biosciences) for 48 h, to facilitate further experimental analyses.
3
Murine Immunology Antibody Panel
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Anti–γ-interferon (IFN-γ), –interleukin (IL)-2, –IL-10, and –IL-17A antibody pairs for ELISA; fluorochrome-conjugated anti-CD4, -CD8, -B220, -Vβ4, -Vβ5, -Vβ8, -CD11b, and -CD11c antibodies for fluorescence-activated cell sorter (FACS); and anti-CD3ε and anti-CD28 antibodies were purchased from BD Biosciences. IL-1β, CCL5, and tumor necrosis factor (TNF)-α DuoSet ELISA kits, CXCL10 antibody pairs, and fluorochrome-conjugated anti–IL-12Rβ2 were purchased from R&D Systems. Fluorochrome-conjugated anti-CD19, -CD25, -CD44, and -CD69 antibodies were purchased from eBioscience, while biotin anti-mouse CD4, in addition to anti-F4/80 fluorochrome-conjugated and live/dead fluorochrome-conjugated antibodies, was purchased from Invitrogen. Anti–P-STAT4 (Y693) antibody was purchased from Cell Signaling, while anti-STAT4 antibody was obtained from Biolegend. The BDC-2.5 mimotope (EKAHRPIWARMDAKK) was synthesized by the University of Alabama at Birmingham peptide synthesis core facility. Xanthine oxidase and β-actin were obtained from Sigma-Aldrich, and r-hIL-2 was purchased from Peprotech.
4
Mouse CD4+ T Cell Isolation and Activation
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Spleens were carefully ground in a 70 μm mesh nylon strainer (Falcon) and rinsed with PBS containing 2% (v/v) FBS to obtain a single-cell suspension. We incubated the centrifuged cell masses with 5 mL erythrocyte lysate on ice for 2 min, followed by a second centrifugation. Subsequently, total CD4+ T cells were isolated from the cell suspension using a Mouse CD4+ T Cell Positive Selection Kit (18,952, Stemcell Technologies). We suspended cell masses in 1 × 10^8 cells/mL PBS containing 2% (v/v) FBS and 1 mM EDTA and then added 50 μL/mL rat serum and 50 μL/mL Selection Cocktail for 5 min. Subsequently, 30 μL/mL magnetic bead solution was added for 3 min, followed by 1.5 mL PBS containing 2% FBS and 1 mM EDTA. The round-bottom test tube containing the mixture was then placed in a magnet for 3 min, then the cells were washed twice with the previously described PBS solution. The purified cells and magnetic beads were left in the test tube for further use. The purified cells were then cultured in Rosewell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin (HyClone), and stimulated with a combination of 5 μg/mL plate-bound anti-CD3ε and anti-CD28 antibodies (BD Biosciences). Chemical inhibitors included BI8626 (HY-120204, MCE), N-Butyldeoxynojirimycin (NB-DNJ, UGCG inhibitor, HY-17020, MCE), and DIDS (HY-D0086, MCE).
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