7 replies to this topic

[B.B.]

Hi,

Odd question - but hear me out!
I'm currently testing the effect of some peptides on the migration of 3T3 fibroblast cells over a scratch wound.
I need to incubate my cells in DMEM media containing the peptide (which is purchased as a powder) for a couple of hours before assaying my cells.

The concentrations of the peptide in the media is really quite small (i.e. 100uM) and so I'm measuring out miniscule amounts of the peptide powder to dissolve into the media - which is leading me to believe that I'm losing some accuracy along the way.

An obvious solution would be to dissolve more powder into a larger aliquot of DMEM media = less room for error.
But I won't be using the peptide/media combination that often and I don't want to just throw it away when my media naturally degrades as the peptide is very expensive.

So I would naturally want to freeze my DMEM/Peptagon combination (in aliquots to thaw out when and as needed).

BUT - Can you freeze-thaw DMEM and it still work optimally?
I can't find anything to say you can't - so I'm guessing it's OK....

[rhombus]

B.B., on 07 February 2012 - 08:28 AM, said:

Hi,

Odd question - but hear me out!
I'm currently testing the effect of some peptides on the migration of 3T3 fibroblast cells over a scratch wound.
I need to incubate my cells in DMEM media containing the peptide (which is purchased as a powder) for a couple of hours before assaying my cells.

The concentrations of the peptide in the media is really quite small (i.e. 100uM) and so I'm measuring out miniscule amounts of the peptide powder to dissolve into the media - which is leading me to believe that I'm losing some accuracy along the way.

An obvious solution would be to dissolve more powder into a larger aliquot of DMEM media = less room for error.
But I won't be using the peptide/media combination that often and I don't want to just throw it away when my media naturally degrades as the peptide is very expensive.

So I would naturally want to freeze my DMEM/Peptagon combination (in aliquots to thaw out when and as needed).

BUT - Can you freeze-thaw DMEM and it still work optimally?
I can't find anything to say you can't - so I'm guessing it's OK....

Dear B.B

My take is that:

If DMEM was "freezable" it would be delivered frozen like FCS/FBS

Your protein/peptide will degrade by multiple freeze-thaw events....so how do you allow for this in your experiments?

Sorry this does not help you very much.....my advice would be to make up the peptide daily and take the hit on the cost

Kindest regards

Uncle Rhombus

[almost a doctor]

For the first time ever I have to disagree with Rhombus on the DMEM issue for various reasons.

1. I used to grow cell lines transfected with differentiation factors (M-CSF and GM-CSF) and will store the media conditioned this way at -20C until use for macrophage or dendritic cell differentitation.  M-CSF was prepared in DMEM and GM-CSF in RPMI, and freeze and thaw did not show a decrease of the differentiation activity.  However I'll add this conditioned media to fresh DMEM or RPMI, so the actual DMEM and RPMI that had been frozen and thawed was only a fraction (usually between 5-20%) of the total media.
2. For a different project, growing leishmania parasites,  we used to prepare our own media. This was DMEM based plus extra aminoacids and nutrients. We used to prepare this at a 2x stock, in 10L and freeze in 500ml bottles and store at -20C for years.  Never saw a difference in parasite growth rate between media at day 0 or media at day 365.   We did not freeze/thaw the media though.
3. Regarding the fact that media is not shipped frozen, I think is actually easier for companies to store and ship reagents at RT or 4C, so the less things that need freezing the better. I think FBS/FCS, antibiotics, and glutamine are stored frozen for longer stability (and in the case of FBS/FCS might even prevent contamination). However, this is just a thought, and I have no actual evidence for it.

Having said all this, I do agree with Rhombus that your protein/peptide will degrade wit multiple freeze/thaw cycles and is actually this that will worry me more than the DMEM. So if you decide to try this I would actually prepare the peptide in a higher concentration you need and then aliquot for freezing so that you only need to thaw once (the time you are going to use it).  
Alternatively, do you have any info on the solubility and stability of your peptide.  Could you dissolve it at a high concentration in a different solvent than DMEM, freeze this way, and then thaw and add to the DMEM (yes, you'll be diluting your media with the addition of the other solvent, but say you prepare it at 100x, the difference in DMEM final concentration will be minimal)

[scolix]

dissolve the whole powder in DMEM, then aliquot in small volumes, like 5ul. Then store them in -80C. Kind of like, use and throw each aliquot. No need to freeze thaw the same tube.

[B.B.]

Hey guys,

many thanks for your comments.

Perhaps I didn't make it clear but I would essentially be making a large volume of the peptide/media mix; aliquoting this mixture; freezing these down and then thawing out the required aliquot when needed - so just freezing and thawing once as 'almost a doctor' suggests

So the "one round" of freeze-thaw of the 'peptide/media mixed aliquot' should be fine.

Just to be on the safe side I will research a little more into the solubility of my peptides and see if I can find a different solvent to dissolve them in to.

[scolix]

Any solvent, there will still be one freeze thaw cycle. Changing the solvent might still not solve your problem.

[bob1]

I would make it at a higher concentration and then dilute it in fresh DMEM, though this doesn't get around the freeze/thaw of the peptide problem.

[leelee]

How about buying some DMEM powder?

You could dissolve your peptide in water (more concentrated than you need, say 2x) and freeze.

Make a 2x DMEM solution with your powder, filter and store.

Thaw peptide, add to 2xDMEM powder (1 to 1), then add to your culture?

Just a thought?