Example 2
CAR-T constructs in pLenti6.3/V5-DEST were purified using the PureLink HQ plasmid purification kit (Life Technology). CAR-T plasmids were lipofected into 293-FT cells with ViraPower packaging plasmids (Life Technologies) according to the manufacturer's protocol. After 48-72 hours, cell supernatant containing live Lentivirus was harvested. Optionally, the virus was concentrated using Lenti-X Concentrator (Clontech), according to the manufacturer's protocol.
Jurkat E6.1 cells were grown in RPMI (Sigma), 10% foetal bovine serum, 2 mM L-glutamine
Jurkat E6.1 cells were transduced for 48-72 hours with viral supernatant in a 50:50 mix of HEK cell supernatant:Jurkat medium: cells at a final concentration of 5×105/ml.
A non-CAR-T construct containing the open reading frame of the Green Fluorescent Protein (GFP) was included as a control. Note that this construct gives cytoplasmic expression.
