Stable expression in HeLa cells and MEFs by retrovirus infection

For stable expression in HeLa cells by retroviral infection, HEK293T cells were seeded in 6-well plates (~3.5 cm in diameter) to 5-10% confluency in DMEM+10% FBS (Day 0).

Day 1
Solution A
Opti-MEM   200 uL
pCG-gag-pol   0.9 ug
pCG-VSV-G   0.9 ug
Retroviral plasmid (pMRX-IP-Halo-rLC3B)   1.8 ug

Solution B
Opti-MEM  200 uL
Lipofectamine 2000   2 uL

Mix Solution A and Solution B
10 min* at r.t.

HEK293T cells (10-20% confluency)
Remove DMEM medium
Add 800 uL of Opti-MEM
After 10-min*, the mixture of the Solution A and B was added to the HEK293T cells.
Incubate for 4-5 h at 37'C in CO2 incubator.
Remove the Transfection Solution and add 2 mL of DMEM+10% FBS.
Incubate for 48 h at 37'C in CO2 incubator.

Day 2
HeLa cells were seeded in 6-well plates (~3.5 cm in diameter) to 5-10% confluency in DMEM+10% FBS.

Day 3
The retrovirus-containing medium (~2 mL) was harvested, filtered with a 0.45-um filter unit (Millipore, Ultrafree-MC).

HeLa cells (10-20% confluency)
Remove DMEM medium.
Add 400 uL of DMEM+10%FBS (+1.2 uL of 8 mg/ml polybrene).
Add 800 uL of the retrovirus-containing solution.
Incubate for 24 h at 37'C in CO2 incubator.
Then, selection was performed with 1 ug/mL puromycin (Sigma-Aldrich, P8833) for 2-3 days.