I would like to treat PBMCs with cancer cell lines conditioned medium (CM) to observe its effect on PBMCs.
So far, I have treated cell lines with 10 micro g/ml in serum free RPMI (FBS limits MMC activity), away from the light, for 3 hours at 37 C and then washed them with PBS x 3. Finally seed cells in complete media to secret CM for 24h.
After several experiments I found that mitomycin C (MMC) treatment may decrease the efficiency of CM and the extracted CM does not have the expected potential for the effect on PBMCs.
I would appreciate if you tell me whether it is necessary to treat cell lines with MMC to get CM.
If No, why?
If yes, what concentration is required? for how many cells (or for how much media)? and for how long?
Thank you
I use a lot of conditioned media from different cell lines for different experiments (for induction of a migratory response, to add some growth factors or to analyze effect of expressed factors on other cells) and I never use any mitomycin. I just centrifugate the conditioned media, after gathering it from over the cell culture, for 5 min. at 400 g to eliminate any possible loose and still alive cells. Then I aspirate the conditioned medium into a new vial, leaving some at the bottom as to not take any possible cells. Mitomycin is used to stop cells from proliferation, so if you transfer any cells together with conditioned medium they would not overgrow your treated cell culture, but centrifugation does it quicker and easier, you just need to be careful with a pellet.
Hi Alaleh,
This may turn out to be a controversial matter, so use your own judgement here!
I don't see the point in treating tumor cells with mitomycin before producing conditioned media, unless you are specifically investigating its effects (or perhaps the effect of the mitomycin-resistant cells). It is not a surprise that your CM have a lower efficiency. Mitomycin is toxic to the cells, and decreased viability will impact in the secretion of factors to the CM. It is also a new source of error, and will add variation to each batch of conditioned media.
If there is no good argument for keeping it in the experiment design and it is affecting your results, I believe you could safely leave it out. I would like to hear someone else's opinion on this.
Good luck with your experiments! Please, share whatever news you have on the matter.
I use a lot of conditioned media from different cell lines for different experiments (for induction of a migratory response, to add some growth factors or to analyze effect of expressed factors on other cells) and I never use any mitomycin. I just centrifugate the conditioned media, after gathering it from over the cell culture, for 5 min. at 400 g to eliminate any possible loose and still alive cells. Then I aspirate the conditioned medium into a new vial, leaving some at the bottom as to not take any possible cells. Mitomycin is used to stop cells from proliferation, so if you transfer any cells together with conditioned medium they would not overgrow your treated cell culture, but centrifugation does it quicker and easier, you just need to be careful with a pellet.
Hello
i agree with Patrycja Koszalka 100%
Kindly look at this link :
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453835/
Good Luck
Thiago, you're definitely right. For this reason, I did this method, which I learned that using MM would result in all the energy of the cells spend to release, due to stopping proliferation. But after searching, I did not find any scientific reason.
Patrycja according you, my flowcytometery results showed a lot of dead cells, in addition to the low differentiation of desired cells.
Saleh, thank you very much for the helpful paper.
Thank you all for your incredibly useful comments, I will aware you from my future experiment result with the correct method.
Best,
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