Cell culture basic questions - Basic questions on cell culture (Jun/18/2008 )
As you guys probably know that I am a newbie in the cell culture field, I would like to ask some stupid questions regarding cell culture.
1) I am doing infection on cell lines. I know starvation is necessary to somehow synchronise the cells growth. I usually do 2 hours starvation. I read some papers stating starvation can be up to 48 hours. I am just wondering, what is the optimal starvation period before infection? Is it an important step in the experiment? How so? Dont the cells cant function properly if long starvation is performed?
2) I am using DMEM media. I realised after a month, the fresh media turned into darker red solution. I guess the glutamine (one of the growth factors) is degrading. Is it important to change the media regularly? or let me put it in a simple question. How long can we keep an opened fresh media bottle?
Thanks!! 
I can't answer your infection question but..
glutamine is an amino acid that breaks down quickly (hence why you add it to media). I usually add fresh glut after 2 weeks or so...but not sure what others do.
Clare
1) I am doing infection on cell lines. I know starvation is necessary to somehow synchronise the cells growth. I usually do 2 hours starvation. I read some papers stating starvation can be up to 48 hours. I am just wondering, what is the optimal starvation period before infection? Is it an important step in the experiment? How so? Dont the cells cant function properly if long starvation is performed?
2) I am using DMEM media. I realised after a month, the fresh media turned into darker red solution. I guess the glutamine (one of the growth factors) is degrading. Is it important to change the media regularly? or let me put it in a simple question. How long can we keep an opened fresh media bottle?
Thanks!!
do you mean synchronizing the cell cycle? just for interest, why is it necessary for infection to synchronize cells?
1) I am doing infection on cell lines. I know starvation is necessary to somehow synchronise the cells growth. I usually do 2 hours starvation. I read some papers stating starvation can be up to 48 hours. I am just wondering, what is the optimal starvation period before infection? Is it an important step in the experiment? How so? Dont the cells cant function properly if long starvation is performed?
Starvation and sync. is not necessary for transfection/infection.
2) I am using DMEM media. I realised after a month, the fresh media turned into darker red solution. I guess the glutamine (one of the growth factors) is degrading. Is it important to change the media regularly? or let me put it in a simple question. How long can we keep an opened fresh media bottle?
Obviously that depends upon the expiry dates of each conponent that you add. The closest expriy date among all component would be the one for your complete media (all components added) too. Another concern is that the longer you store a media after opening/constituting it, more are the chances of contamination. So, entire subject then becomes subjective to your handling, your freeze's opening/closing frequency, sanitation, temp maintenance etc.
Thanks!!
As cellcounter mentioned, you are not starving your cells for the infection/transfection. You serum starve cells so that they synchronize in GO (quiescence). When you release the cells (by adding media with serum) all the cells re-enter the cell cycle at the same time. This is a good method for investigating cell cycle. How long you need to starve your cells in order to obtain synchronization depends on the cell line but two hours will never be enough. Generally it's always going to need to be 24-48 hours (my CHO cells are always 48hrs). Now, I used to serum starve cells for 2-4 hours in order to quench certain signaling pathways. Then, after drug treatment, I could assess what effects the drug had on a specific signaling pathway. As for your media, I wouldn't worry. I've used a bottle of media for months without any ill effects. Media will always change color after being opened for awhile but as long as the media returns to a normal color after about 10 mins in the incubator, it's ok. Some people add extra glutamine because it does tend to break down but I've never bothered because my cell lines aren't that sensitive. You need to check with people who are working with the same cell lines or look at the ATCC website to see what they recommend.
The colour change is due to a pH change: there isn't enough CO2 dissolving into solution from the air, resulting in a high pH and the phenol red going from red (neutral) to purple (basic). This is normal and shouldn't be a problem so long as the colour returns to red when put into a CO2 incubator.
ok, I think your stock medium changes color to deep pink, right? in that case it is because of oxidation. do not open/close your stock so much. aliquot your media.
usually when you grow cells in incubator the media change color to yellowish after some time. that's another issue.
as for infection, it's better not to starve,....if you do that your cells are not in a normal condition and your lysates are not standard....do not starve....I do infection with a virus too,.....but we decided not to starve the cells anymore, I guess you do that because you don't want them to proliferate anymore right?....so better to increase the amount of virus you use instead of starving your cells.
Wow, I have so many replies from you guys. You guys are just so great! Thank you very much!
For my case, I usually starve it to stop the growth cause for bacteria infection, one need to have roughly 80% of confluency so that the bacteria will have some space to move around. I am not sure about virus though. Dont you have to starve it as well?
Does it make a difference for starving and non starving cells? If not, why all the people in my lab is starving the cells? By starving, what are the advantages besides stopping growth. I guess it is very important if one is studying the cell cycle.
Thank you so much for the input!