Developing a Titration Kill Curve ( G418 , Hygromycin B and Puromycin )
Split non - transduced , confluent cells 1 : 5 in 10 ml DMEM + 10 % FBS media .
Transfer 0.5 mL cell suspension into 24 - well plate containing 500 µl of ( media + drug ) .
- G418 Sulfate
- Hygromycin B
- Puromycin Dihydrochloride .
Examine viability every 2 days .
Culture for 14 days .
Replace the media containing antibiotic every 3 days .
Use the lowest concentration of your antibiotic that begins to give massive cell death in 3 days and kills all the cells within two weeks .
A general starting point is usually 400 mg / ml G418 for HeLa cells and 200 mg / ml hygromycin for CHO cells .
In mammalian cells the optimal level of puromycin is typically around 1 mg / ml .
HeLa cells are often selectable with 500 mg / ml G418 , 500 mg / ml hygromycin , or 2.5 mg / ml puromycin , and SHSY - 5Y cells are often selectable with 600 mg / ml G418 or 200mg / ml hygromycin .
