Stable transfection - When to start selection? (Aug/09/2005 )

Hi,
I am performing stable transfection with Lipofectamine. The manufacturer tells you to wait for 3 days after transfection to split cells and start selection.
I had about 80-90% confluency when transfected. Now, 24 hours later, the cells are completely confluent. I am afraid that they will not like to be still 2 days in such a crouded way.
So should I split them and still wait 2 days before selection, or should I split them and start selection right away? or should I let them be still for 2 days(i'm afraid not this one)?

Heeeelp!

coco

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hi
with my cells i start at 60% confluency and apply selection after a spilt 3 days after.
if cells are confluent, split them at least 3 times and start selection.

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Split when your cells are confluent - do not wait the three days.

And I think this is what Fred means ... before feeding with selective media, split your cells so they will take another 3 days to become confluent so your selective agent has time to kill the untransfected cells before it is metabolised.

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hi
you're right. I was unclear
the selection occurs in most cases on active cells

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day1: 110000 adherent cells for example for the inoculum in P35mm (6 well plate), day2: 16h00 post inoculum you have 30-50% confluent cells and you make transfection (=t0)
t=48h00 trypsine cells put them 2000 cells in a P100, keep others cells in a flask and start the selection with the appropriate agent G418 (800µg:ml), 15 days after you could have clones than you can pick up using cylinders,

important tips:
to make nice clones , i rinse 4 times with pbs and let the trypsin do its jobs for 5 mn only ,and you have single cells
i notice when your cells are confluent you have few selection effect so when i make stable transfection i control this and trypsinise them when i have 30% confluence

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