Problem with HeLa not attaching to six well plates - (Aug/07/2007 )
Hi. In the last two weeks I have been having problems with my "adherent" cells. They are not attaching to all wells of six well plates. Actually they grow only in one or two wells which appear to be always the same. I have already tried plates from three different lots, and the problem persists. I tried 48-well plates and I am having the same problem. I tried petri dishes and sometimes they do attach and sometimes not. This has never happened to me before. I must add that the cells anyway keep growing nicely in the T-75 flasks. I thought the line was already too old, so I thawed a new culture from the deep freezer, but it is behaving exactly the same. Has this happened to anyone else? Any ideas? Thanks.
which hela cell type are they ? standard hela are good adherent cells, HeLaS3 are very weak adherent cells
also check the composition of medium. You need Calcium chloride in it to enhance cell attachment.
also check the composition of medium. You need Calcium chloride in it to enhance cell attachment.
They are standard HeLa. My medium is DMEM plus horse serum and antibiotics. It contains Calcium chloride. Before these last two weeks the cells have been attaching nicely with the same medium. Plus, as I mentioned the cells are attaching very well to the T75 culture flask. Thanks. Any other idea or suggestion?
new serum stock? or new serum serie ?
did you apply particular treatment ? did you check for mycoplasma contaminations of your equipments?
did you apply particular treatment ? did you check for mycoplasma contaminations of your equipments?
The bottle of serum we used for the media was thawed a month ago, the cells were giving no problems with this bottle of serum. I will discard it and try a new one thanks. How do you test for mycoplasma? And, if there is contamination with mycoplasma, I should treat all instruments? Also add something to the incubator?
many posts in this forum tells to throw away cultures in case of contamination.
detection of mycoplasma
http://www.protocol-online.org/forums/inde...showtopic=24898
http://www.protocol-online.org/forums/inde...showtopic=21660
useful too
http://www.protocol-online.org/forums/inde...showtopic=10342
http://www.protocol-online.org/forums/inde...?showtopic=8380
Did you make a new bottle of DMEM media for these cells.
Do you add glutamine or does the DMEM come with glutamine? If you miss out on glutamine, the cells can behave odd too.
Is it a new batch of 6-well plates? This may be too simple, but the plate must be tissue culture treated for the cells to attach, if an order mix up or by accident you got the non-TC treated plates that could be the problem...
Do you add glutamine or does the DMEM come with glutamine? If you miss out on glutamine, the cells can behave odd too.
There is a person in the lab in charge of preparing the DMEM, and then each of us takes his own bottle and supplements it with antibiotics, serum and glutamyl-alanine for regular use. So yes, my bottle has glutamine. Thanks a lot guys, I will test for mycoplasma, and see if that is the problem. Any other ideas are always welcome!
Thanks beccaf22. I tried three different lots of 6-well plates. All the same model I have used before (Costar 3516) without any problems for a couple of years.