293FT Cells for Transfection

293FT lentivirus packaging cells (90% confluent on 6-cm tissue culture dish)

Complete cell growth medium

Gelatin (0.1% in H₂O)

Dulbecco's phosphate-buffered saline (DPBS), no calcium or magnesium (Life Technologies 14190-144)

Trypsin-EDTA (Life Technologies 25300-062)

1. Pipette 1 mL of gelatin into three 6-cm dishes. Swirl to coat the bottom of each dish then aspirate off the gelatin. (This coating helps the cells adhere to the surface during transfection.)
2. Tilt the lids slightly and leave the dishes in the tissue culture hood partially covered until the gelatin dries completely (∼15 min).
3. Aspirate the medium from the 293 cells. Gently add 1 mL of 1× PBS to wash away any remaining medium from the cells. Aspirate the PBS.
4. Add 1 mL of trypsin-EDTA to the cells. Place the dish in the 37°C incubator for 2–3 min.
5. Gently tap the side of the dish to dislodge the adherent cells. Transfer the trypsin-EDTA and cells into a 15-mL conical tube.
6. Add 5 mL of complete medium to inactivate the trypsin and mix gently. Centrifuge at 1000 rpm for 2 min to pellet the cells.
7. Aspirate the medium from the cells and replace with 4 mL of fresh complete medium. Resuspend the cell pellet by triturating with a 10-mL pipette until all clumps are dispersed.
8. Add 3 mL of fresh medium to each of the three gelatin-coated dishes plus one extra dish.
9. Add 1 mL of resuspended cells to each dish and rock the dishes gently in all directions to evenly distribute the cells. Incubate the cells at 37°C with 5% CO₂ for 24 h. (This 1:4 split should provide ∼1–2 × 10⁶ cells per 6-cm tissue culture dish after 24 h. Cells in the three gelatin-coated dishes can be used for transfection. Cells may be maintained in an uncoated dish for later use.)