How to select suspention tranfected cells

[Laleh Kamalian]

Hi. I am a new member to this site and a PhD student. I am working with cells which grow in suspension. I have managed to transiently transfect my cells using a plasmid vector and Lipofectamine. Now I am ready to try to make the stable transfection. However I haven't got a clue how to select the stably transfected ones using G418, especially how to separate the dead ones when the massive death occurs and how to pick up a single clone!! I have already started the G418 optimization. Can anyone help me please.
Laleh

[gandalfthegrey]

Hello Laleh,
I have done stable cell selection with suspension culture and its tricky.

I have used a similar protocol here:

Stable Cell Selection of Suspension Cultures Protocol

1. Transfect your cells.
2. Culture your cells with several passages (1-3) in a T-75 flask with selection agent (e.g. Use hygromycin, neomycin or similar).
3. Make sure you optimize your selection concentration using death curves or similar.
4. Collect and then pellet your cells carefully (low rpm) and resuspend at a concentration of 1 x 10^5 cells/ml in your selection media.
5. Then aliquot 1 drop/well of cell suspension in to each well of a 96-well tissue culture plate.
6. NOTE: Make sure to maintain 2-10 plates for each transfection you do.
7. Check your growth plates every day.
8. If you get a cell population growing in a well, subculture the cells by diluting them 1:10 with selection media and seeding.
9. Continue subculturing the cells in this manner for several more passages (5-10) to insure that a clonal population is established.

Based on protocol here:
Selection of Transfected Suspension Cells

If you have a different protocol please post it here to share.
Thanks!

[Laleh Kamalian]

Hi.
Thanks for your reply. I have come across similar protocols myself but it seems to take a long long time. There are other options like using semisolid a media. I am still thinking and searching about it. I will post the result when I got a good one.
LK