Conditioned media question - (Aug/02/2009 )

Hi people,

I want to investigate the effect of conditioned media on the behaviour of adherent cells. I need to grow my cells in high-serum media (10% FBS) for 5 days and then I need to add serum-free media to the same dish for 3 days. The idea is that I am trying to create conditioned media by letting the cells secrete different factors in the serum free media for 3 days. However, as I mentioned, before adding the serum-free media the cells were grown for 5 days in 10% FBS media. I do 3 long washes with PBS before changing from 10% FBS to serum-free media, but I am still worried that some serum proteins will bind to the dish/plasma membrane of the cells and will then be released into my serum-free media.

After 3 days, I isolate my conditioned media and add it to different cells. I can observe that the conditioned media exerts siginificant effect on the behaviour of these cells compared to controls (controls are fresh serum-free media). However, similar behaviour may be observed if these cells are grown in high serum media. So I am not sure if the results I got are due to the fact that my cells indeed conditioned my serum-free media by secreting certain factors in it, or if alternatively some serum components/proteins from the 10% FBS media have been unspecifically bound to the plasma membrane of the cells and later released into the serum free media.

Do you think it is possible that after 3 washes in PBS, when changing from 10% FBS to free serum media, there is still significant amount of serum proteins left on the plasma membrane of my cells - enough to be secreted in the serum free media and still be active 3 days later? What method do you recommend to use to be sure that I get rid of any residuals from the 10% FBS and not damage my cells? I was thinking something like 3xPBS wash and then 2h pre-incubation in serum free-media before adding fresh serum-free media to be conditioned. Do you think this will be enough?

I would really appreciate you advices and thanks in advance!

I believe there is a way to estimate the amount of residual liquid trapped between cells with radioactively labeled sucrose. So my suggestion is if you can demonstrate that the effect you saw from the conditioned medium is greater than that from the residual serum can do ( even with some extra amount).

However, it is entirely likely that the protein factors could have some affinity to cell surface or matrix between cells and can not be washed away. I guess to demonstrate that you would need much sophisticated analytical measurement, and may include protein purification from conditioned medium.

Biolog1 on Aug 2 2009, 10:47 AM said:

may be observed? is it similar or not. this statement is ambiguous.

just set up the proper controls...for your concerns, the best negative control should be cells that are washed 3X in PBS and incubated in fresh serum-free media for 1 day or less such that any residual from the FBS that might contribute to the effects you observe after 3 days will be detected. essentially, if you see an effect with 3 day conditioned media but not 1 day conditioned media then you can be reasonably confident that secreted factors are accumulating.

to support this, you can then do the experiment again, but this time boil the conditioned media that you collect. this should denature most of the protein factors that accumulate, such that the 3 day conditioned media that was having an effect, should no longer do so.

you can also filter your conditioned media to remove or retain protein factors of a desired MW. this would shed some light on whether or not the factors are peptides or larger secreted/ectodomain shedded transmembrane proteins.