Hi !
I plan to carry out some live-cell imaging of HeLa cells. Our microscope has a 37°C heating device but no %CO2 regulation.
Also, I'd like to image some red fluorophores (mCherry, Alexa568), so I'd like to use a medium without Phenol red.
I can't find any company selling CO2-independent, phenol red-free DMEM. Does anyone know where to find such media ?
I know that addition of HEPES (±10 mM, to phenol red-free DMEM) could help to buffer, but for how long ? Imaging could take more than an hour.
Thanks for you help !
Edit: Is OptiMEM a CO2-independent medium ?
This might be more expensive than other options, but Gibco makes custom media and ordering it is very easy.
They have a proprietary CO2-independent formulation that I'm sure they could make without phenol red.
Leibovitz L-15 medium may be worth a try. It is a medium formulated and buffered by phosphates and free base amino acids instead of sodium bicarbonate. This medium is designed for supporting cell growth in environments without CO2 equilibration.
Good luck
Dear Nicolas,
As far as I am aware, OptiMEM is not CO2 independent.
The problem with just adding HEPES to a commercial formulation is that it already contains Na Bicorbonate at a level for 5-10% CO2. So you will still get a pH shift. Instead, I make a Hank's BSS without phenol red that is buffered for use in air. Some people in the lab have also used it for imaging neurons, since we have a similar microscope incubator problem, and not all cells like L-15 (I don't know about HeLa).
I have never tried using the same proportion of Na Bicarbonate/HEPES to make up a phenol-red free DMEM from powder, but it might be worth a try.
The recipe is
Hank's BSS salts (w/o phenol red) powder to make 1 L
1 mM Sodium Bicarbonate
5 mM HEPES
If you have never made media or BSS from powder, what you do is start with 900 mL of TC quality water and dissolve the salts/media. You then add the sodium bicarbonate and HEPES (make sure they dissolve), and pH the solution. You then bring the volume to one liter. Sterile filter the solution into sterile bottles in the tissue culture hood and store at 4 C as normal.
Since we work on neurons, we also add extra glucose to this mix before adding the buffer components. I am not sure you will need to do that.
Since cell metabolism will also contribute to a change in pH, I suggest you take some of the mixture you decide to use, add some phenol red, put it on a dish of your cells in the incubator, and see how long it takes to get a pH shift.
Good Luck!
Jill
Thank you all !
1. Alberto: I asked for a quote at Gibco, and I am now waiting for their price.
2. Vehid: Thanks for the suggestion, it looks like the fastest way to get the things done !
3. Jill: Thanks for your comprehensive reply ! In case HeLa don't like L-15, I'll try your method !
Nico
Edit: HeLa should like L-15 (http://himedialabs.com/TD/AT011.pdf)
It’s great that HeLa like L-15! Definitely the easiest way to go. We use it all the time.
I wish you great images.
Jill
An update,
Yes HeLa grow in L-15. Do they like it, probably not enough. I haven't measured their growth precisely but they definitely don't grow as fast as in DMEM. Plus, their morphology is totally different (much more elongated and not so "triangular/square").
For the moment, we will stick to CO2-independent medium that we have (with phenol red). I did not receive any reply for the quote I asked to Gibco. I'll try again...
I will also check Jill's option !
Nico
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