2 replies to this topic

[ltaggart]

I have been making stable cell lines using TransIT, transfecting in a GSK3S9A plasmid in a pcDNA vector in three different cell types: SKOVs, MCF-7s and CaOV3s.  The process worked really well for the SKOVs and they are growing fine however, for the other two cell lines whenever I got to the stage where I was passing them for the first time after antibiotic (G418) selection they won't settle back onto the new plate and in the case of the MCF-7s they formed huge clumps of cells.  So now I have several plates of floating cells that won't adhere.  It's not a problem with the plates as other cells are sticking to them and I have been using the same trypsinisation technique since I started cell culture and have never had a problem.  
I was wondering if anyone else has experience this or know why this seems to be happening and how I can fix it? I was thinking it could be something to do with the harsh environment growing in G418?

[cellcounter]

ltaggart, on Jul 15 2009, 09:42 AM, said:

I have been making stable cell lines using TransIT, transfecting in a GSK3S9A plasmid in a pcDNA vector in three different cell types: SKOVs, MCF-7s and CaOV3s.  The process worked really well for the SKOVs and they are growing fine however, for the other two cell lines whenever I got to the stage where I was passing them for the first time after antibiotic (G418) selection they won't settle back onto the new plate and in the case of the MCF-7s they formed huge clumps of cells.  So now I have several plates of floating cells that won't adhere.  It's not a problem with the plates as other cells are sticking to them and I have been using the same trypsinisation technique since I started cell culture and have never had a problem.  
I was wondering if anyone else has experience this or know why this seems to be happening and how I can fix it? I was thinking it could be something to do with the harsh environment growing in G418?

Floating cells are more likely to be dead. Did you do trypan blue on them?

Three possibilities come to mind.

1. Excessive trypsinization - easy to correct.
2. Excess G418 - Do a kill curve for these cells first, then use the minimum amount of G418 that gives 100% cell death.
3. If this is a problem with your test plasmid only, and not empty vector, then you have a killer gene/loss of adherence gene on hand.

And I hope you are using the transfectino reagent according to manufacturer's guidelines.

[Reis.V.P]

cellcounter, on Jul 16 2009, 08:08 AM, said:

ltaggart, on Jul 15 2009, 09:42 AM, said:

I have been making stable cell lines using TransIT, transfecting in a GSK3S9A plasmid in a pcDNA vector in three different cell types: SKOVs, MCF-7s and CaOV3s.  The process worked really well for the SKOVs and they are growing fine however, for the other two cell lines whenever I got to the stage where I was passing them for the first time after antibiotic (G418) selection they won't settle back onto the new plate and in the case of the MCF-7s they formed huge clumps of cells.  So now I have several plates of floating cells that won't adhere.  It's not a problem with the plates as other cells are sticking to them and I have been using the same trypsinisation technique since I started cell culture and have never had a problem.  
I was wondering if anyone else has experience this or know why this seems to be happening and how I can fix it? I was thinking it could be something to do with the harsh environment growing in G418?

Floating cells are more likely to be dead. Did you do trypan blue on them?

Three possibilities come to mind.

1. Excessive trypsinization - easy to correct.
2. Excess G418 - Do a kill curve for these cells first, then use the minimum amount of G418 that gives 100% cell death.
3. If this is a problem with your test plasmid only, and not empty vector, then you have a killer gene/loss of adherence gene on hand.

And I hope you are using the transfectino reagent according to manufacturer's guidelines.

Hi

I have the same problem using my HepG2 transfected with RNAi. When i thawed them, they can't adhere in the flasks..... Don't know what happens, my freezing and thawing protocols are Ok. I use G418 to select my cells with FuGene delivery system. One thing you can try is to increase bovine serum and avoid to use G418 after thawing cells, we use to start selection after 2 passages when they attached ( when they did... )

Regards

Reis,V.P.

Edited by Reis.V.P, 19 July 2009 - 06:52 AM.