TITLE: Culture and transfection of HEK293T cells
AUTHORS: Hanqin  Li, Dirk Hockemeyer

[DESCRIPTION]
This protocol describes a standard procedure culturing and transfecting HEK293T cells

Protocol overview:
A. Culturing HEK293T cells
B. Transfection of HEK293T cells with Lipofectamine 2000

[STEPS]
SECTION: A.	Culturing HEK293T cells
1. HEK293T cells are cultured in HEK293T medium in 10 cm dishes.
SECTION: A.	Culturing HEK293T cells
2. Passage cells when the culture reaches 80% confluency.
SECTION: A.	Culturing HEK293T cells
3. Remove medium
SECTION: A.	Culturing HEK293T cells
4. Wash once with 5 ml DPBS
SECTION: A.	Culturing HEK293T cells
5. Add 1 ml 0.25% Trypsin with EDTA, tilt and shake the dish so that the Trypsin covers the entire dish.
SECTION: A.	Culturing HEK293T cells
6. Incubate at 37 °C for 1 min

SECTION: A.	Culturing HEK293T cells
7. Add 5 ml fresh HEK293T medium to inactivate Trypsin
SECTION: A.	Culturing HEK293T cells
8. Pipet 10 times to dissociate the cells and mix
SECTION: A.	Culturing HEK293T cells
9. 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. 
SECTION: A.	Culturing HEK293T cells
10. HEK293T cells usually needs to be passaged every 2 days. 
SECTION: A.	Culturing HEK293T cells
1.1. HEK293T Medium
     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 100mM      5 ml         
 
SECTION: B.	Transfection of HEK293T cells with lipofectamine 2000
11. One day before transfection, dissociate HEK293T cells with Trypsin as described above
SECTION: B.	Transfection of HEK293T cells with lipofectamine 2000
12. Seed 250,000 cells/1 well of 12-well plate
SECTION: B.	Transfection of HEK293T cells with lipofectamine 2000
13. Change to fresh medium 1 h before transfection 
SECTION: B.	Transfection of HEK293T cells with lipofectamine 2000
15. 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 5 min.

SECTION: B.	Transfection of HEK293T cells with lipofectamine 2000
16. While waiting, in micro centrifuge tube II, add 125 µl Opti-MEM and 250 ng total plasmid. Mix by pipetting.
SECTION: B.	Transfection of HEK293T cells with lipofectamine 2000
17. Mix tube I and II by gently pipetting 3 times. Incubate at Room temperature for 20 min. 

SECTION: B.	Transfection of HEK293T cells with lipofectamine 2000
18. Mix one time by gently pipetting. Transfer all transfection reagents into one well of a 12-well plate, drop-wise. 
SECTION: B.	Transfection of HEK293T cells with lipofectamine 2000
19. After adding the reagent to all wells, shake the plate to mix. 
SECTION: B.	Transfection of HEK293T cells with lipofectamine 2000
20. Culture in 37 °C incubator 20 min.

SECTION: B.	Transfection of HEK293T cells with lipofectamine 2000
21. Change to fresh medium and culture for another 2 days. Collect samples or passage once if longer culturing is needed. 
SECTION: B.	Transfection of HEK293T cells with lipofectamine 2000
14. Label two micro centrifuge tubes as I and II