PBMCs were collected under informed consent from healthy donors. PBMCs were stimulated with 600 U/ml IL-2 (rIL-2; R&D Systems, Minneapolis, MN) and 50 ng/ml anti-CD3 (OKT-3; eBioscience, San Diego, CA) in AIM-V medium (Life Technologies, Carlsbad, CA) supplemented with 2% human AB serum for 7 days. The concentration of rIL-2 was increased to 1,000 IU/ml on day 8.
Electroporation were performed with the Nucleofector device II (Lonza, Cologne, Germany). 10 × 106 PBMCs were activated for 8 days as described above and were thereafter re-suspended in 100 μL of Cell Line Nucleofector Solution V (Lonza, Cologne, Germany) and TCR mRNA was added at 200 μg/ml47 (link). The mixture was placed in a certified cuvette (Lonza, Cologne, Germany) and electroporated. After electroporation, cells were re-suspended in AIM-V medium (Life Technologies, Carlsbad, CA) supplemented with 2% human AB serum and 100 IU/ml rIL-2. Transfected cells were maintained in a humidified 37 °C and 5% CO2 incubator until flow cytometry analysis and/or co-culture experiments.
Electroporation were performed with the Nucleofector device II (Lonza, Cologne, Germany). 10 × 106 PBMCs were activated for 8 days as described above and were thereafter re-suspended in 100 μL of Cell Line Nucleofector Solution V (Lonza, Cologne, Germany) and TCR mRNA was added at 200 μg/ml47 (link). The mixture was placed in a certified cuvette (Lonza, Cologne, Germany) and electroporated. After electroporation, cells were re-suspended in AIM-V medium (Life Technologies, Carlsbad, CA) supplemented with 2% human AB serum and 100 IU/ml rIL-2. Transfected cells were maintained in a humidified 37 °C and 5% CO2 incubator until flow cytometry analysis and/or co-culture experiments.
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