We've been trying to transduce THP-1 cells with some P2X7 constructs (Blasticidin resistance gene also present) using Lentivirus and Blasticidin selection. This has worked well in HEK293T cells before but doesn't seem to be working in THP-1s as after Blasticidin addition all the cells die within a brief period. Not just that, the GFP control transduction doesn't seem to have worked either.

Protocol:

• Day 0: Plate 1mL THP-1 cells with 8ug/L Polybreene in 6-well plate and add 1mL lentivirus. Centrifuge for 1.5 hours at 1800 RPM at 32C (Spinfection).
• Day 2: Replace media with media containing 10ug/mL Blasticidin
• Day 4: Split cells through multiple passages so that the dead cells are filtered out.

Some ideas I had was perhaps the Blasticidin concentration is too high or we add it too fast, killing too many cells at once, causing the remaining live cells to die as well? Since the virus has previously been tested and had worked in HEK293T cells I doubt it is that, but we have also made new viral stocks.

Any thoughts?