The production and use of lentivirus should be done in appropriate biological safety cabinet. And lentivirus should be disposed after treatment in 10% bleach.
Day 1: Early afternoon (11-12AM)
- Use 293T cells of around 70-80% confluency grown in DMEM high glucose + 10% FBS, 1% Glutamax and 1% penicillin–streptomycin to plate the cells for the transfection.
Cells to be used should be healthy and of low passage number. Cells should reach 90-95% confluency after 24 hours for transfection. (Growth medium without antibiotics can be used for the plating, however, it may or may not affect the efficiency of transfection.) 1 million 293T cells can be plated with 2 ml medium in 6 well plate to obtain 95% confluency the next day. The number of cells to be used should be optimized.
Day 2: 11 AM
- In one tube (A);
250ul opti-mem medium + 7ul Lipofectamine 3000 (Mix well by quick vortexing)
In next tube (B);
250ul opti-mem medium + 6 ul Lipofectamine enhancer + 1.5ug plasmid + 0.3ug PMD2G + 1.2ug PSPAX2
- (Order of addition can be started from smaller volume to larger volume for proper mixing) and mix well by quick vortexing.
- Transfer the mixture in tube A to tube B, QUICK VORTEX, and SHORT SPIN and incubate at room temperature for 15 mins.
- Remove 1ml growth medium (half medium) from the plate.
- Gently transfer the mixture in tube B to the cells from the side of the plate dropwise, gently rocking the plate simultaneously.
- Incubate the plate in 37 degree Celsius incubator with 5% CO2 incubator.
After 5-6 hrs. ,
Change the medium by removing old medium and replace with fresh warm growth medium. Incubate overnight.
Day 3: 24 hrs. post transfection
- Check GFP to calculate the transfection efficiency.
- Harvest the virus. Collect the medium in a sterile tube and store it at 4 degree Celsius and replace with new fresh, warm growth medium.
Day 4: 52 hrs. Post transfection
Harvest the second round of virus. Collect the medium and mix it with the harvest from 24 hrs.
Virus concentration: Viral concentration can be done to get high titer virus using ultracentrifugation.
- Filter the collected virus containing medium through 0.45um filter.
- Centrifuge using a SW-28 rotor @ 25,000 rpm, 90 min, and 4C.
- Discard the supernatant and dissolve the viral pellet with PBS or serum free medium and store at -80 degree Celsius. Avoid multiple freeze thaw by storing in smaller aliquots.