We cultured all cell lines in RPMI 1640 medium with 2.05 mM L-Glutamine (Hyclone) supplemented with penicillin (100 μg Ml−1), streptomycin (50 μg mL−1) and 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO2 and checked periodically for mycoplasma contamination using MycoSensor qPCR Assay Kits (Agilent Technologies, Santa Clara, CA, USA). We cultured primary lymphoma cells RPMI 1640 medium with 2.05 mM L-Glutamine supplemented with penicillin (100 μg mL−1), streptomycin (50 μg mL−1), 10% human serum (Sigma-Aldrich, St. Louis, MO, USA), 10 μg mL−1 IL-2 and 10 μg mL−1 IL-7 (PeproTech, Rocky Hill, NJ, USA). Prior to drug treatments, the cells were stimulated for 48 hours with 10 μg mL−1 plated anti-CD3 (clone HIT3a) and 2 μg mL−1 soluble anti-CD28 (clone CD28.2) both from Thermo Fisher (Waltham, MA, USA)
Soluble anti cd28 clone cd28.2
Manufactured by Thermo Fisher Scientific
Sourced in United States
Soluble anti-CD28 (clone CD28.2) is a laboratory reagent used in immunological research. It is a monoclonal antibody that binds to the CD28 receptor, which is expressed on the surface of T cells. The anti-CD28 antibody is soluble and can be used to stimulate T cell activation and proliferation in cell culture experiments.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd3 clone hit3a, by Thermo Fisher Scientific (2 mentions)
Human serum, by Merck Group (1 mentions)
Interleukin 2 (il 2), by Thermo Fisher Scientific (1 mentions)
Interleukin 7 (il 7), by Thermo Fisher Scientific (1 mentions)
Mycosensor qpcr assay kit, by Agilent Technologies (1 mentions)
Ficoll paque plus gradient, by GE Healthcare (1 mentions)
Cd45ro apc cy7, by BioLegend (1 mentions)
Cd3 fitc, by BioLegend (1 mentions)
Cd4 bv605, by BioLegend (1 mentions)
Facsdiva software, by BD (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
2 protocols using soluble anti cd28 clone cd28.2
1
Primary lymphoma cell culture protocol
2
AhR Modulation of T-cell Activation
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De-identified blood obtained from healthy donors (Stanford Blood Center, Stanford, CA) was fractioned by a standard centrifugation protocol using a Ficoll-Paque® PLUS gradient (GE Healthcare Biosciences, Pittsburgh, PA). The buffy coat layer was isolated and washed in HBSS containing 2% BCS.
Cells were seeded into 24-well tissue-culture-treated plates at 1 × 106 cells per well and stimulated with plate bound anti-CD3 (clone HIT3a) and soluble anti-CD28 (clone CD28.2, both at 1 μg/mL and from Thermo Fisher Scientific) for 18–24 h. Cells were treated with AHR antagonist (CH223191) or agonist compounds pyocyanin, 1-hydro-phenazine, FICZ, TCDD, or DMSO vehicle for 24–48 h. Cells were harvested for gene expression at 24 h and flow cytometry analysis at 24–48 h. For sorting of CD4+ CD45RO+ and RO- T cells, freshly isolated human PBMC was stained with CD3-FITC, CD4-BV605, and CD45RO-APC-Cy7 (Biolegend, San Diego, CA) and acquired using FACS Aria cell sorter and FACS Diva software (BD Biosciences, San Jose, CA). Sorted cells were treated with AHR ligands for 48 h and analyzed as described above.
Cells were seeded into 24-well tissue-culture-treated plates at 1 × 106 cells per well and stimulated with plate bound anti-CD3 (clone HIT3a) and soluble anti-CD28 (clone CD28.2, both at 1 μg/mL and from Thermo Fisher Scientific) for 18–24 h. Cells were treated with AHR antagonist (CH223191) or agonist compounds pyocyanin, 1-hydro-phenazine, FICZ, TCDD, or DMSO vehicle for 24–48 h. Cells were harvested for gene expression at 24 h and flow cytometry analysis at 24–48 h. For sorting of CD4+ CD45RO+ and RO- T cells, freshly isolated human PBMC was stained with CD3-FITC, CD4-BV605, and CD45RO-APC-Cy7 (Biolegend, San Diego, CA) and acquired using FACS Aria cell sorter and FACS Diva software (BD Biosciences, San Jose, CA). Sorted cells were treated with AHR ligands for 48 h and analyzed as described above.
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