my cell line doesnt change the colour of the media - even when cells are 100 % confluent (Sep/28/2009 )

Hi all, we have developed cell lines from mouse ovaries and testis which we are characterising at the moment.

They were immortalised using the pRITA (reversible immortalization with TAg) plasmid nar.oxfordjournals.org/cgi/content/full/32/18/5529 with gene-juice carrrier.

I have noticed a strange thing about them (as has my colleague who has alos immortalised a male cell line with same plasmid/carrier)

The cells when confluent dont change the colour of the growth media-dmem/f12 with phenol red, even when they have been confluent for a few days

The media is fine as it changes colour when used with other cells we are culturing but not on these two cell lines.

Does anybody have any experience in seeing this or can explain why it happens?

I understand the phenol red is an indicator used to chage purple(ish) when exposed to alkaline environments and orange(ish) in acidic but we dont see this.

So the only answer can be the cells do not change the pH of the growth media but how can this be?,

They grow at a decent rate with doubling time of just over 24 h and as I said have been grown to over 100 % confluency but still no colour change. the fact that both cells have been immortalised the same way and both seem to not reduce the pH it must be the pRITA plasmid that controls this but i dont know how.

Any suggestions would be great.

Cotchy.

very interesting. Do the cells remain alive for a long period after they have become confluent?
The only things I can think of is that either the cells stop growing due to the cell-cell interaction or they require some sort of hormone stimulation to be metabolically active. They may be remaining in the rest cycle possibly waiting for an activating signal?

What media are you using?

If you subculture the cells when confluent, do they grow just fine again in the new flask until confluent again?

Stephan on Sep 29 2009, 07:01 AM said:

I have tried not to leave them too long after confluency but sometimes over weekends they can become very confluent (estimated 130 - 150 % confluent) and they all are fine and survive when sub cultured. The cells are always dividing and always seem to be metabolically active

I am using DMEM/Hams F12 supp with 0.5 % penn/strep and 5 % FCS

I also use the exact same media (from the same aliquots) for my other immortalised cells (immortalised using pSV3-neo) and they dont have this affect as they turn the media orange quite soon after or during confluency.

I usually use 14-15 ml media per T75 for these other cells but for the cells that dont change the media colour 10 ml per T 75 always seems to be enough even as I said over weekends.

Maybe its a case that my other cells turn the media orange quite quickly and this is what I am accustomed to.

So now these other cells dont have this effect as quick and maybe it could be quite normal to other researchers, however cell counts over a weekend of culture estimate the number to be around 6 million +, so surely they would have an effet on the phenol red.

As i said in my last post, the growth rates for all my cells are quite similar from around 20 - 24 h doubling time

I think you may have something when you talk about what you are accustomed to. Cell lines may be very different to each other as far as metabolism is concerned. Because you immortilized these cells yourself, there aren't many references you can consult. As an experiment I would leave a confluent flask with minimal media until either 1) the cells die and the phenol red is still red or 2) the phenol red eventually changed colour. If number 1 is true then I am stumped. Maybe you have created the ultimate cell line - that which does not require media! Heh, maybe you're actually wasting money - just grow them in PBS!

P.S. - why are you only using 5% FBS? I get the high pen/strep due to primary tissue origin but maybe the FBS is running out and the cells don't become metabolically active. during the test i mentioned above increase the FBS to 10% or once confluent spike the media with more FBS to raise it to 10%. - im interested to know the answer to this!!

CellSpecific.com on Sep 30 2009, 07:24 AM said:

Cells have been grown at same time in same incubator after seeding from same aliquots of media but no colour change, i suppose when i say no change there is a slight change after time but not like with the other cells.

The FBS at 5 % is just something i have done from the start (I took this conc. from paper on same cell type i pressume i am working with) with all cells, and as i perform alot of cytotox assays such as MTT and neutral red I would have to reduce the FCS to 5% for these so as I seen the fact they grew well and the negative controls in MTT and NR (in just media with 5% FCS) always seem to be metabolically active I had no real reason to increase the concentration.

I have a falsk of 80 % confluent cells in incubator now they have been in the media a couple of days and i will leave them unil next week just to see if any change then.

CO2 works fine on the incubator with the sensor serviced recently.

I am sure the media will eventually change colour its just the fact it seems to take so long compared to my other cells, i will also dperform a cll count using trypan blue on cells next week to see if still viable and of course get anidea of how many cells are there.