CAUTIONARY NOTES OR SPECIAL CONSIDERATIONS
Concentrated lentiviral preparations should be tittered on each target cell type prior to usage as viral titer values vary by cell type/line.
Researchers should have completed basic laboratory safety, chemical safety, bloodborne pathogens, and biosafety level-2 (BSL-2), training prior to conducting this protocol.
Day 1: Prepare Lentiviral Transfection Plates
1. Prepare a 0.0004% solution of poly-L-lysine (1:25 dilution if using the poly-L-lysine solution described in the attached materials section) in water and filter sterilize using 0.22 μm filter.
2. Apply poly-L-lysine solution to each plate and incubate at room temperature for at least 2 hours (optimally 3-4 hours), agitate to re-distribute solution occasionally.
a. NOTE: Apply 10 mL of poly-L-lysine solution for 15 cm plates.
b. NOTE: Apply 4 mL of poly-L-lysine solution for 10 cm plates.
Day 1: Prepare Transfection Mixes in 50mL tubes
1. Warm appropriate cell culture medium, PBS, and Opti-MEM in 37 °C water bath.
2. Aspirate the poly-L-lysine solution from each plate and rinse three times with ddH2O immediately before adding cells and transfection mix. Do not allow the plates to dry.
a. NOTE: Rinse with 15 mL for 15 cm plates and 10 mL for 10 cm plates
b. NOTE: Aspirate third rinse of ddH2O immediately prior to cell plating in Step 7.
3. Prepare appropriate volume of transfection mixes in separate 50 mL tubes as indicated in the attached table.
4. Incubate Mix 1 and Mix 2 separately for 5 minutes.
5. Combine Mix 1 and Mix 2 and incubate for an additional 20 minutes.
6. During the 20-minute incubation:
a. Follow steps 2-13 of the section “Sub-culturing cells” of the SOP for 293T splitting
b. Resuspend cells to a concentration of approximately 2.0 x 106 cells/mL in growth medium.
7. In a 15cm plate add the following (in order):
a. Fresh growth medium
i. NOTE: Add 6.75mL for 15cm plate or 2.75mL for 10cm plate
b. DNA-Lipofectamine 2000 complexes in Opti-MEM
i. NOTE: Add 6.75mL for 15cm plate or 2.75mL for 10cm plate, the mix by swirling.
c. 293T cells at approximately 2.00 x 106 cells/mL
i. NOTE: Add 13.5mL for 15cm plate or 5.51mL for 10cm plate, then mix by swirling.
8. Incubate in a humidified incubator at 37 °C with 5% CO2 for 24hrs.
Day 2: Replace 293T growth media
1. In the morning (~9:00 AM) aspirate the transfection medium into a flask containing bleach.
2. Gently add fresh growth medium to each plate of transfected cells.
a. NOTE: Add 25mL for 15cm plate or 12mL for 10cm plate
3. Incubate cells in a humidified incubator at 37 °C with 5% CO2 for 48 hours
Day 4: Collection of Viral Particles
1. Begin at approximately 10:00AM.
2. Centrifuge the conditioned medium at 1000 x g in a tabletop centrifuge for four minutes to pellet the majority of cellular debris
3. After centrifugation, filter the supernatants through 0.45 μM pore syringe filters to remove small particulate debris.
4. Add 11 mL of each viral supernatant to ultracentrifuge tubes (two tubes for the filtered supernatant from each 15 cm plate).
a. NOTE: Use balance to weigh viral supernatant in ultracentrifuge tube. Fill remaining ultracentrifuge tubes with ddH2O and use for balances. (Make sure similar weight)
5. Spin at 50,000 x g (20,000 RPM in a Sorvall TH-641 rotor) for 2 hours and 20 minutes at 20 °C.
6. Carefully decant the supernatant.
a. NOTE: Treat it with bleach prior to disposal.
7. Resuspend the pellet in the medium of the target cells to be transduced by vigorous triturating.
a. Resuspend concentrated viral particles from a single plate with 1 mL of target cell growth medium.
i. NOTE: Resuspend in 1mL when using either 15cm or 10cm plates
8. Transfer the supernatant(s) to new tube(s).
9. Pellet insoluble material at top speed in a microcentrifuge for two minutes.
10. Aliquot viral supernatant into cryotubes (to ensure proper biosafety, these tubes must have an internally-threaded O-ring seal) as 50 μL aliquots and store at -80 °C.
a. NOTE: Aliquot volume may vary.