Culture and transfection of HEK293T cells

HEK293T cells are cultured in HEK293T medium in 10 cm dishes.

HEK293T Medium

A	B
DMEM, high glucose	385 ml
Fetal Bovine Serum (FBS)	50 ml
L-Glutamine (100X)	5 ml
MEM Non-Essential Amino Acids (100X)	5 ml
Sodium Pyruvate 100 mM	5 ml

Final volume: 500 mL

Passage cells when the culture reaches 80% confluency.

Remove medium

Wash once with 5 ml DPBS

Add 1 ml 0.25% Trypsin with EDTA, tilt and shake the dish so that the Trypsin covers the entire dish.

Incubate at 37°C for 0h 1m 0s

Add 5 ml fresh HEK293T medium to inactivate Trypsin

Pipet 10 times to dissociate the cells and mix

Transfer 1 ml of cell suspension to a new dish pre-added with 9 ml HEK293T medium. Shake to mix well.This is a 1:6 splitting.

HEK293T cells usually needs to be passaged every 2 days.

One day before transfection, dissociate HEK293T cells with Trypsin as described above

Seed 250,000 cells/1 well of 12-well plate

Change to fresh medium 1 h before transfection

Label two micro centrifuge tubes as I and II

In micro centrifuge tube I, add 125 µl Opti-MEM and 6 µl Lipofectamine 2000. Mix by gently pipetting 3 times. Incubate at Room temperature for 0h 5m 0s.

While waiting, in micro centrifuge tube II, add 125 µl Opti-MEM and 250 ng total plasmid. Mix by pipetting.

Mix tube I and II by gently pipetting 3 times. Incubate at Room temperature for 0h 20m 0s.

Mix one time by gently pipetting. Transfer all transfection reagents into one well of a 12-well plate, drop-wise.

After adding the reagent to all wells, shake the plate to mix.

Culture in 37°C incubator 0h 20m 0s.

Change to fresh medium and culture for another 2 days. Collect samples or passage once if longer culturing is needed.