Forward transfection in 6-well plate
1. Seed hESCS in 6-well plate and culture for 12-24 hours.
2. Prepare transfection mixture as follows:
1. Tube-1: 100ul Opti-MEM, 5ul Lipofectamine stem transfection reagent.
2. Tube-2: 100ul Opti-MEM, 2ug plasmid.
3. Mix tube-1 and tube-2, incubate at RT for 10min.
3. Change 1.5ml/well fresh mTeSR1, add transfection mixture onto cells.
4. 24h after transfection, refeed 1ml fresh mTeSR1 without removing old medium.
5. 48h after transfection, passage cells to new 6-well plate, add 0.5ug/ml puromycin and treat for 24h or until non-transfection control cells are all killed by puromycin.
6. Culture with mTeSR1 supplemented with Y27632 for 2 more days, then change to normal mTeSR1.
Reverse transfection in 6-well plate
1. Prepare transfection mixture as follows:
1. Tube-1: 100ul Opti-MEM, 10ul Lipofectamine stem transfection reagent.
2. Tube-2: 100ul Opti-MEM, 2ug plasmid.
3. Mix tube-1 and tube-2, incubate at RT for 10min.
2. Treat cells with Versene or accutase, dissociate cells into singe cell suspension or small clumps (3-5 cells/clump).
3. Seed cells into 6-well plate (5e5/well or 1:5 split).
4. Add transfection mixture onto cells, culture in 37℃ incubator.
5. 24h after transfection, refeed 1ml more mTeSR1 without removing old medium.
6. 48h after transfection, passage cells into new 6-well plate, add 0.5ug/ml puromycin and culture for 24h or until non-transfected control cells are all killed by puromycin.
7. Culture with mTeSR1 supplemented with Y27632 for 2 more days, then change to normal mTeSR1.