Protocols: Lentiviral Infection
Lentiviral Infection of T, B and BM Cells
Media
- C10: Lymphocytes: RPMI 1640 + 10% FCS, L-glu, pen/strep, hepes, NEAA, Na-pyruvate and 2-BME
- D10: Lymphocytes: DMEM + 10% FCS, L-glu, pen/strep, hepes, NEAA, Na-pyruvate and 2-BME
- Bone Marrow: DMEM + 15% FCS, L-glu, pen/strep, IL-3, IL-6, SCF
Day 1
- Plate 0.7x106 293FT cells in a 60mm dish in 4ml of D10 media
Day 2
- In the early evening, begin transfection
- For each infection, place 150 Buffer EC into a siliconized 1.5 mL eppendorf tube.
- Add 0.5ug of PI, PII and PIII packaging vectors. Do not premix packaging vectors.
- Add 1.0ug of viral plasmid (pLL3.7, etc)
- Gently mix
- Add 16ul of Enhancer
- Gently mix, wait 5’
- Add 32ul of Effectene
- Gently mix, wait 5’
- Add to plated 293FT’s from day 1. Gently drip into media.
- Incubate at 37 C.