The ecotropic retroviral packaging cell line, Platinum (Plat)-E cells (Cell Biolabs), were plated (2.2 × 106 cells) in PLAT-E medium (DMEM, 10% FBS, 10 µg/ml blasticidin, 1 µg/ml puromycin, and 100 U/ml penicillin/streptomycin) on 10-mm culture plates. After 24 h, Plat-E cells were transfected with DNA encoding the desired TCR and/or construct using the Effectene transfection kit (Qiagen). At 48 h, medium was replaced with T cell medium (IMDM, 10% FBS, 2 µM l -glutamine, 100 U/ml penicillin/streptomycin, and 25 µM 2-β-mercaptoethanol). On days 2 and 3 after transfection (at 72 and 96 h), virus-containing supernatant was collected. Splenic T cells isolated from P14 Thy1.1+ mice were stimulated with α-CD3e (clone 145-2C11) and α-CD28 (clone 37.51; both from BD PharMingen) and IL-2 (50 IU/ml aldesleukin; University of Washington Pharmacy) and transduced with retroviral supernatant by spinfection in polybrene (5 µg/ml, 90 min at 1000 g) 24 and 48 h after T cell activation.
α cd28 clone 37.51
Manufactured by BD
α-CD28 (clone 37.51) is a lab equipment product. It is a monoclonal antibody that binds to the CD28 receptor on T cells.
Automatically generated - may contain errors
3 protocols using α cd28 clone 37.51
1
Retroviral Transduction of Murine T Cells
2
Analyzing Cytokine Production in Vaccinated Mice
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Spleens from vaccinated mice were collected at the indicated timepoint, mechanically disrupted, and treated with ACK (Ammonium-Chloride-Potassium) buffer to lyse erythrocytes. Splenocytes were cultured at 37 °C, 5% CO2 in RPMI media with 10% FBS and 1.25 µg/mL each α-CD28 (clone 37.51, BD) and α-CD49d antibodies (clone R1–2, BD). MHCI-specific ovalbumin SIINFEKL/OVA257–264 or MHCII-specific ovalbumin peptide OVA323–339 were added at 2 µg/mL (Invivogen, San Diego, CA, USA). Phorbol 12-myristate 13-acetate (Promega) and ionomycin (Tocris, Bristol, UK) were added at 1.25 µg/mL and 0.02 µg/mL, respectively. After 24 h, interferon-gamma (IFN-γ) in the supernatant was quantified using the mouse IFN gamma ELISA (AbCam, Cambridge, UK).
3
T Cell Activation and Functional Assays
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Splenocytes were dissociated into single cell suspensions and treated with ACK lysis buffer as above. Remaining leukocytes were resuspended at 2.5 × 107 cells/well in 5 ml of RPMI with additives (see cell culture methods section) and were activated overnight with α-CD3ε (clone 145-2C11) (500 ng/ml) and α-CD28 (clone 37.51) (500 ng/ml) (BD Bioscience) with recombinant human IL-2 (rhIL-2) (100 U/ml) and mouse IL-7 (mIL-7) (2 ng/ml) in 5% CO2 at 37°C. The cells were harvested the next day and centrifuged at 400 g for 4 minutes to remove activating antibodies. Cells were then resuspended in RPMI with additives including rhIL-2 (100 U/ml) and mIL-7 (2 ng/ml). Functional assays and immunophenotyping were performed on days 5 to 7 post activation.
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