I have to treat my cells with environmental pollutant. Is it necessary treat the cells in serum free media ?. Do we need to seed our cells in serum free media as well??.
Hi Dhirendra - to answer your question properly I would need to know how you plan to analyse the cells (secretion factors, apoptosis measurements, ab's etc).
In general though you would treat any cell line (and controls) in full media (+serum) to make them as "happy" as possible. Also if you add adherent cells to TC plates/wells in the absence of serum they will not adhere (the FBS contains serum proteins such as Fibronectin that sticks to the plate/wells - the cells stick to these proteins). Note: this is not the case for a nod-adherent line obviously,.
If you do need to seed an adherent line into serum free media you will have to coat the wells with an ECM protein prior to seeding.
Alternatively (f the test recommends serum free media) add the cells to the wells (so that they will be sub-confluent the next day - the exception to this is intestinal cells such as CaCo2), allow to adhere overnight then change the cells to serum free media after attachment.
If you could give some idea as to the test(s) you aim to perform I might be better able to answer the question.
If in doubt I would do both (+/- serum).
Hope this helps,
Gary
Hi
I agree with Gary, he provided lots of useful information. I would just add that some serum compounds can interfere with your drug, e.g. drug can be bound to serum albumin or other carriers, affecting its transport into the cell and therefore its effects, ...
Moreover, In order to minimise the influence of serum and still keep the cells "happy", you can use artificial serum or serum replacements - ITS, for example.
So you should consider the relevance of +/- serum media, and, as suggested - if in doubt, use both.
Have luck with your experiments.
Jakub
Agree with above. Serum often interfere assays by scavenging test compounds and affect the basal activities. For assays with short incubation time, I usually run it in serum free condition (e.g. Calcium assay in HBSS). For assay that require more than a few hours of incubation, serum may be critical for the attachment or survival of the cells. In most cases. I would plate cells in complete medium and then change to serum-free or reduced serum medium at or right before stimulation. In assays that are very sensitive to serum, I place cells in reduced serum medium (e.g. UltraMEM with 0.5% serum) and then change to serum free (UltraMEM alone) during the assay. Those defined medium can help the cells to attach and survive even in serum-free conditions.
Preferably and appropriately, the cells should be seeded in plates using their complete growth media, while treatment should be prepared in serum-free (non-phenol red, depending on the assay) media. Subsequently, the initial complete growth media in the plates should be aspirated just before treatment.
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