I am selecting BaF3 and CCRF-CEM cell line with G418. It had been almost 2 months. I am not able to get rid of dead cell. After 2 months also I am still seeing dead cells with alive cells. These cells are in culture with complete RPMI media with G418. I did also kill curve to know the appropriate concentration of G418 for respective cell lines. Please help me with this. Thank you so much.
Hi Nitesh, hope you're fine!!
1) Can you please report the % of dead cells that are present in your cell population after 2 months of selection? If the fraction of dead cells is low it could be perfectly normal.
2) Are you growing cells in presence of growth factors, such as IL3 for Baf ?
Best,
Rocco
Hi Rocco,
I am good. I hope you're doing well also,
1) My cells are in 100 ml now (alive-1 million cells per ml). I have 50 % dead and 50 % alive cells. In addition, I had also performed a qPCR from these cells last week and I got overexpression of inserted vector in comparison with the empty vector.
2) I am using IL3 in complete RPMI media for BaF3 cell line.
The vector is a pMSCV Neo retroviral vector. I used normal retroviral infection method and after 4 spins I had kept the cells in the complete RPMI media for a week and started selection with G418 with appropriated concentration.
In order to get rid of dead cells I tried two different strategies:
1) Changing media after 3 days. So dead cells will disappear while centrifugation or changing media during subculture (Not worked)
2) Changing media only once a week, so that alive cells will take over the dead cells.
Till now I had kept cell very dense. So I am thinking to subculture them in right density. Do you think it is right option?
Or
It’s better to repeat the infection again?
Thank you so much,
With Regards,
Nitesh
You should try to keep them at best concentration, something around 200-400k/ml. I would also test the presence of mycoplasma...
Is it possible that your overexpressed target is toxic? did you find a similar behaviour in your control cell line?
Best,
Rocco
Yeah, I am using the lowest G418 concentration which can kill untransfected cells.
So you are suggesting me to decrease the concentration of G418 what I am using now?
Control cells grow much slow, than target gene.
I will try to do Mycoplasma test.
Thank you,
With Regards,
Nitesh
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