The transduction efficiency depends upon the target cells and experimental procedure.
Day 1: Plate cells
1. Plate 2–10 x 10^4 of the target cells per well in a 24-well plate 24 hours prior to viral infection. Use 0.5 ml of cell specific medium supplemented with 5% heat-inactivated fetal bovine serum, and penicillin-streptomycin (optional) for each well. Incubate the cells at 37°C with 5% CO2 overnight.
Note: Make sure the cells reach 70-80% confluent at the time of transduction. Actual cell number to be plated depends on the cell types.
Day 2: Transduce target cells
2. For each well, prepare 0.5 ml of virus suspension diluted in complete medium with Polybrene at a final concentration of 5–8 μg/ml.
Note: Use several dilutions of pseudoviral stock (0.001 ul to 10 ul). In addition, we recommend including a transduction with the eGFP control and other appropriate positive and negative controls. Mix the virus with the medium gently by inverting the tubes several times. Do not vortex.
3. Infect the target cells by removing the old culture medium and replacing it with 0.5 ml of diluted viral supernatant. For one well (mock well control), add 0.5 ml of complete medium with Polybrene. Place the plates in a 37°C incubator with 5% CO2 and incubate cells overnight. (Optional: Place the plates for 2 hours at 4-8°C; then transfer the plates to a 37°C incubator with 5% CO2 and incubate cells overnight.)
Note: Incubating cells with lentivirus for 2 hours at low temperatures can significantly increase the transduction efficiency. But it may be omitted if the cells cannot tolerate low temperatures.
Day 3: Replace medium/Split cell culture
4a. Replace the old medium with 0.5 ml of fresh complete medium (without Polybrene). Continue to 5a.
4b. Or split the cells 1:5 to 1:25 depending on the cell types by trypsinizing and re-seeding the cells onto 6-well plates or 10-cm culture dishes. Continue incubating for 48 hours in cell specific medium. Continue to 5b.
Day 5: Analyze transduced cells or start drug selection of stably transduced cells
5a. The infected target cells can be analyzed for transient expression of transgenes using an appropriate biological assay. If you have used an internal eGFP control, determine the percentage of infected cells by counting fluorescing cells by flow cytometry or with a fluorescent microscope.
5b. To select stably transduced cells, replace old medium with fresh complete medium containing the appropriate selection drug every 3–4 days until drug-resistant colonies become visible (generally 7–14 days after selection).
For more tips on lentivirus production and titering, visit GeneCopoeia website.