I have the XTT salt (powder) and I would like to use it for XTT assay.
For that reason, I am going to follow the Roche CellProliferation Kit which constitues of:
- XTT in RPMI 1640 medium,1 mg/ml, filtered through 0.2 μm pore size membrane (I am going to use DMEM instead of RPMI since I don't have RPMI in my lab right now)
- PMS (N-methyl dibenzopyrazine methyl sulfate) 0.383 mg/ml (1.25 mM) in PBS, filtered through 0.2 μm pore size membrane
The kit is meant to be stored at at -15 to - 25°C.
I plan to prepare the XTT and PMS solution as described. Do you think it's ok to prepare more of them than just for a single use and to freeze the solutions? The XTT solution contains cell medium which raises my doubts. Is it possible that the manufacturer of the Roche Proliferation Kit puts something more in the solutions (cryopreservative?) that is not mentioned in the composition?
I attach the Roche Proliferation Kit leaflet file.
I would be grateful for any responses. Thank you.
I highly doubt it. Frozen media works fine after thaw, there is nothing in there that would cause issues. The only thing that might change is pH.
I have done this with MTT, but I froze the MTT at a very high concentration so that I only add like 10uL or so per well. In this case, you shouldn't have to worry about using media in the frozen storage, I just use PBS.
Other thing to double check, I'm pretty sure you need media that doesn't have phenol red.
Shen-An Hwang Thank you for your answer! Yes, I will use no-phenol red medium to avoid altering the xtt result.
Dear Paulina,
in my opinion, you can prepare the XTT and PMS solution as described and it is ok to prepare more of them than just the volume for a single use and to freeze the solutions? Maybe this can help to safe time (even if this is not so much).
However, freezing could potentially reduce efficacy of your solution/measurement due to reduced signal intensity - as described by the purchaser (maybe minimally). If you want to compare results of different measurements, I would suggest to follow the protocol of the kit / manual, which is often designed to get best possible results (it should be optimized in this kit!).
However, it is your choice, both should be possible (preparing fresh solution for each experiment or use a frozen stock.
Bist wishes.
I would like to ask one more question: is freezing media in general OK? For example, if I have the DMEM bottle that I know I would not finish before the expiration date - can I freeze it, e.g. in alliquots? I searched this topic in the internet and I found that there is no clear advice. Some people freeze media, some don't. Some say that repeatable freezing and thawing cause precipitations.
What is your experience? Can the sealed / unsealed medium bottle be frozen? Can medium be frozen in smaller volumes?
Dear Paulina,
freezing the the medium alone should be no problem. However, including supplements this could influence opimal use of the medium.
Since DMEM is quite unexpensive, I would propose to prepare freh medium when it is needed instead of freezing rather ole medium for moth or years.
- Badges
- Science method