A549 cells not doubling - has anyone been through this with other cell lines? (Jun/02/2010 )
My cells are not doubling! I've tried everything: spiking up to 15% FBS, adding more media than usual, placing a pan of water in the incubator for extra humidity. It seems that every few passages they just give up on dividing and just sat there, not dying but not really living either. I mean what's the point of living when you're not doubling, especially when YOU'RE A CANCEROUS CELL! I'm really frustrated... has anyone been in the same situation, or have any idea what else I should try?
Hmmm, it is a bit worrying that you can't get A549 to grow. Can you give a bit more detail about your cultures (medium, tempreature, %CO2 etc.), How old are your cultures (i.e. how long have you had them up for)? Have you looked for contamination?
I would do a DAPI stain to check for possible contamination with mycoplasma, had a similar problem recently and mycoplasma seemed quite likely to be the problem, as it may alter cell cycle progression induce cell death alter gene expression...
The conditions are: 5% CO2, rel. humidity varies from 26 to 66, now it's ~26 since I took out the water pan. I didn't think it made any difference. I use Nunc flasks. I usually take them out from LN2 and immediately thaw in 37C for about 2mins, dilute with media, and leave in T25 overnight. Up until this point the cells look healthy, and they fill up the flask in less than 24hrs. I would then transfer to T75, and this is when the cells refuse to grow. Sometimes they would grow fine in T75 for 2-3 passages, then they stop. I use DMEM with high-glucose, + L-glutamine. When I passage, I wash with HBSS, then trypsinize for 5mins in 37C, add complete DMEM, then centrifuge at 500 x g for 5 minutes. There's been two contaminations in the past, but was promptly eradicated. I haven't checked for mycoplasma contamination yet. I think we have a PCR-based kit, but is there a quicker way of detecting it? How do I go about doing the DAPI stain?
butterfingers on Jun 7 2010, 08:55 AM said:
Hi! I also think that Mycoplasma could be the reason. There is a very quick and not too expensive kit from Lonza (MycoAlert). You only need a plate reader and around 20 minutes.