Protocols_CellCulture_Lentiviruses and MOI Calculation - BackmanLab/BackmanLab.github.io GitHub Wiki
Lentivirus Packaging & MOI Calculation
Preparing viruses:
-
Make sure the packaging cell 293T is at low passage (<P10). The lower the better.
-
Day 0: 1 day before transfection, plate 293T cells into wells of a 12-well plate. Aim for ~50-60% confluency for the next day.
-
Day 1: Before transfection, bring FuGene HD to RT before use.
-
Dilute total amount of 1ug DNA in 50 ul OptiMEM (per well).
-
For lentivirus packaging, mix the following amount of DNA: 0.5 ug viral vector+0.45 ug dR8 + 0.05 ug VSVG
-
Add 3 ul FuGene HD dropwise and incubate 5 min at RT.
-
Add FuGene & DNA mix onto the cells.
-
Day 2: After ~24 hr, change the media into fresh DMEM.
-
Day 4: ~60 hr after transfection, collect the viral supernatant. Spin 5 min at 4000 rpm to pellet cell debris.
-
Filter the supernatant through a 0.45 um syringe filter or 0.45 um low protein binding membrane (Millipore Steriflip-HV, 0.45 µm, PVDF, Cat no: SE1M003M00) for larger viral supernatants.
-
Aliquot into tubes of 200 ul virus and freeze them at -80C.
-
After freezing, use one of the aliquots to measure the viral titer (each freeze-thaw will decrease viral titer so be careful and consistent).
Calculating percent transduction and MOI:
-
Day 1: 1x10^5^ cells per well were plated into a 12-well plate in the appropriate standard media with 8 ug/ml polybrene.
-
Each well received a different titrated virus amount (usually 50-100 ul) along with a no-transduction control.
-
Day 2: After the cells were incubated overnight, they were enzymatically detached using trypsin. Cells were counted and each well was split into duplicate wells (you can use 6-well plates).
-
Day 3: Add puromycin to only one replicate. After 3 days (or as soon as no surviving cells remained in the no-transduction control under puromycin), count the cells to calculate a percent transduction.
-
Percent transduction is calculated as cell count from the replicate with puromycin divided by cell count from the replicate without puromycin multiplied by 100. After determining percent transduction, you can calculate MOI as described below:
