How do I culture adherent TZM-bl cells?! - (Sep/18/2007 )
I'm very new to the biochemistry and cell biology front, having just finished a Chemistry masters. So please don't laugh too hard at my question if it's extremely dumb...!
I'm trying to grow up an adherent cell line, the TZM-bl cells. According to all the articles etc that I have it should be fairly straight forward, but they look like they're just about ready to keel over and die.
This is what I did:
1. I thawed a new vial from liquid nitrogen in 10% DMEM on Wednesday.
2. The next day I added some more media (5ml).
3. Last Friday I trypsinised the cells with 0.25% trypsin, washed with PBS and resuspended in fresh media for the weekend and the cells looked fine!
When I checked on them first thing yesterday morning they were only 20% viable and the media in the flask was cloudy (the media stock was still fine, so I figured it wasn't contamination..)
PLEEEEEEZE help!! This was the only vial of TZM-bl stock we had.....!
Telisha (Serial Cell Killer)
If your media is cloudy, my first suspicion would be contamination. Even if your media stock is fine, you could still have contamination, perhaps from your pipette or something....
Can you see anything besides cells and dead cells in the flask?? Try a high magnification and check for moving particles which would be bacteria- they are much smaller than your cells. If you did have bacterial contamination it would look really grainy and very different to just having dead cells.
As far as other types of contamination (eg fungus), I have been fortunate enough to have never had this happen (although now that I've said that, the lab f*%k up fairy will undoubtedly make it happen!!!) but I think you would be able to see some sort of growth besides you cells in the media.
Was your flask confluent when you trypsinised it?? and how many new flasks did you split into?? Maybe your cells have just majorly overgrown and their are heaps of them dead in the media??
Have you still got your flask or did you throw it away???
You can try to order the TZM-bl cells from NIH (aidsreagent.org).
apart from this: they are easy to culture, just like regular HELA cells (from which they are derived). It's probably a contamination, so you have to make 100% sure about using sterile technique.
Can't you practice on a cell line of which you have more vials, or get one that's been cultured in your lab and excercise a bit?
Vairus: The TZM-bl cells I'm using did come from the NIH...
Before starting on these cells I grew up some CEM.SS cells for practise. They are growing like mad and I never had any problems with contamination or cells dying....
Lauralee: I checked the cells for bacterial contamination as you suggested, and yup! There're stacks of small moving particles! Thankfully there's no fungal contamination and the cells still seem to be holding on - just barely..! I washed the adhering cells with 5 x 5ml volumes of PBS to try and remove the baterial contamination, but two days later the contamination was back... I washed them again and added media (with 2% Strep/Pen) to it, but again it wasn't long before the little pests tried to take over. How do you normally get rid of a bacterial infestation like this..?!
The cells were confluent when I first trypsinised them and split them into two flasks with fresh media. I still have both of these flasks (this is where the contamination started) and haven't tried to trypsinise the cells again as there aren't too many of them left alive. What do you suggest I do..?!
PBS-washing will not help you to get rid of the bacteria.
Discard this bottle and try to acquire a new batch of cells. But before doing this: make sure everything you use is sterile (just incubate your medium on its own etc @ 37 for a day and you should notice your contamination if it's in your medium or so).
Thanks vairus. Man, this is going to be painful...!
Thanks for the help guys!