Stable transfection in suspension cells

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Sat Jan 19 15:01:46 EST 2008


Dear Laleh,

you could try this: make a dilution of your cells that you have 5-10
cells per ml. Then seed 100ul in each well of a microplate (a total up
2-5 plates) under selection conditions. You'll get approx 1 cell per
well. Then watch them become more (or not). You might use a medium
containing 50% conditioned medium from 293 HEK (or similar well
growing cells), just take the not too much used medium and filter
through a syringe filter in order to remove any cells. Then you cells
they don't feel so lonely, means they get some growth factors and grow
more easily. Then expand those wells where your cells grow (into 24
well plates) and check aliquots for gene expression. You might re-
check your cells for expression from time to time, as there is some
chance that a) those loosing the transgene overgrow your culture and
b) that you had more than 1 cell in the initial microwell.

If you have access to a cell sorter (FACS), then apply selection
pressure after transfection and collect the propidium iodide negative
cells (adding Ethidium bromide 10mg/ml stock 1:10.000 might do it as
well) for microplate seeding. This probably makes things much easier,
especially when you get a low transfection rate.

Good luck!

Wo



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