WEHI-3BD cell culturing
Posted 11 June 2011 - 06:31 PM
I have a question in regards to WEHI-3BD. In the past, our lab has used RPMI + 10% FCS, 1% PS and 1% Glutamax to culture these cells for IL-3 production.
This year however, we've chosen to use the method as described by Galli's lab (http://gallilab.stan...ments/2WEHI.pdf), which they use DMEM media. But it seems the cells don't seem to grow as well, and somehow they appear much more adherent than usual. It gets to a point where I need to constantly blow the cells using pipettes or taping the flask. And even so they just won't come off. We try to avoid using Trypsin to get them off. The other option I've tried is using PBS to wash the cells, and that seems to work. But we're not sure if that causes the cells to die.
Does anyone have suggestions? I just don't know why the cells are not growing. I'm inclined to believe it's the media.
This is what I've done so far. I took them out of cryofreeze on Wednesday. I saw the cells and they looked rather confluent on Thursday, which is why I've moved them to a T-75 flask. But on Friday, I moved some cells from T-75 to T-175 flasks. I checked on Sunday, they weren't growing. Even in the initial T-75 flask, which I did about a 1 in 2 split.
Posted 12 June 2011 - 03:41 PM
Posted 12 June 2011 - 04:54 PM
Posted 12 June 2011 - 09:55 PM
Posted 13 June 2011 - 04:52 PM
While you are doing the change of medium, you should observe your cells to ensure that they have not changed their morphology and growth rates (and if you are interested in them - expression of your target genes/proteins and controls).
Posted 13 June 2011 - 07:32 PM
you brought up an interesting point. I have always subcultured and maintained the cells in DMEM, and when I seed them, I differentiate my cells for 3 weeks in MEM. What I noticed though as the passage number increases, the cells appear to grow faster, be it the cells subcultured in DMEM or the ones seeded and differentiated in MEM. Morphology wise they look the same under the microscope. Come to think of it, the cells was in MEM when we bought it, and we added it straight to DMEM, so it's possible that the cells are still adapting to the new media thus the increased growth rate.