Hi Nouran, Serum starvation of your cells 24 hours before you start a cell based assay is a common procedure.  The reason its performed is to synchronize all your cells to the same cell cycle phase.  In this regard when you add agents to the cells, theoretically you have removed the impact that cell cycle will have on your cells response to treatment.  However i usually leave cells overnight, not 24 hours.  The reason i reduce the length of time is that cells are dependent on FCS concentrations for active growth.  By removing FCS for long periods it may result in your cells growth being negatively effected.  Therefore i would recommend optimizing the optimum length of serum starvation before you start experiments. 

Hello Nouran, serum starvation is a common step in just about every cell based assay because the composition of serum (FBS) is not well defined and contains a myriad of small molecules, proteins and cytokines. These addition variables can certainly 1) complicate your results trying to delineate if your compound killed the cells or if a reaction of serum and your compound killed the cells, or even if a bad batch of serum killed the cells.  

Even defined serum is designed to induce growth to a certain extent with various proteins.

So, in order to eliminate variables and bring closer together correlation and causation, serum is not used for preliminary cell based assays.

That being said, I do sometimes add an extra group that is

my compound tested plus serum 

No treatment serum starved

No treatment with serum

compound tested on starved cells

(this can help bring more questions and ideas/ analysis) (Also to mimic in vivo work, your drug being tested will never be acting alone, it will be exposed to some aspect of proteins/ molecules found in serum).

- Kyle

I agree with Kyle, this is to limit the variability associated with FBS, but it's also to remove any protective factors that may be in the serum and to sort of starve the cell a bit so it takes up things from the media moreso than it would under non-starved conditions (namely the compound of interest)

Hi Nouran, Serum starvation of your cells 24 hours before you start a cell based assay is a common procedure.  The reason its performed is to synchronize all your cells to the same cell cycle phase.  In this regard when you add agents to the cells, theoretically you have removed the impact that cell cycle will have on your cells response to treatment.  However i usually leave cells overnight, not 24 hours.  The reason i reduce the length of time is that cells are dependent on FCS concentrations for active growth.  By removing FCS for long periods it may result in your cells growth being negatively effected.  Therefore i would recommend optimizing the optimum length of serum starvation before you start experiments. 

I agree with Alex: It's mostly for synchronizing cell cycle. However, different components (like albumin) can also bind your drugs and thus lower the effective concentration why you don't always wish to have serum present even when you do your drug treatments. This, however, very much depends on your cells: I have had cells that were perfectly happy for longer periods of times without serum, some that needed at least 0.1% serum, and others that couldn't go below 1% serum in order to stay alive...

Thanks everyone! You helped a lot. But in my MTTs I did not starve any cells. Does that mean that the results are not reliable? Based on your experience and knowledge, do I need to repeat the experiments?

Thanks a lot guys:)

Yes, you should re-run your assays in the absence of serum. Serum is known to interact with MTT assays to some degree, and some of the components of serum are capable of reducing MTT to formazan.

Additionally, as mentioned above, you want to ensure the components in serum do not interact with your compounds directly and lower their effective concentration, and you want to prevent any growth factors/other protective factors in the serum from acting upon your cells while you're investigating the compounds' effect. Do not be surprised if some of your compounds are much more cytotoxic when you run the assays in the absence of serum.

MTT assays can be run very simply. We used to seed our cells and allow them to attach and grow for 24-48h in normal media with serum (until ~ 60-70% confluence). Then, just remove the medium, and replace with serum-free media containing the compounds of interest (or vehicle). Then, incubate for as long as you wish and run your viability assays. Try to avoid going more than 24 h in serum-free media before running your MTT assays. 

As mentioned before, every cell type is different and may require different parameters (higher/lower seeding densities, low or moderate amounts of serum, etc). Hope that helps!

Yes actually it helped a lot. Thanks a lot :):)

Hi folks, the results you obtain with the serum starved cells might be different to the results you get when you use them logarithmically growing in the presence of serum or when you use confluent, non dividing cells. Principally, serum starved cells should have stopped all signaling that depends on serum factors, so it is believed that signalling events can be better observed without this background.

We have published a study comparing several growth conditions and responses to food derived signals; you might have a look at this: Int J Food Sci Nutr. 2014 Mar;65(2):219-25. doi: 10.3109/09637486.2013.839631. Epub 2013 Oct 11. Cell culture condition-dependent impact of AGE-rich food extracts on kinase activation and cell survival on human fibroblasts.

Best wishes, Norbert

Besides the reasons mentioned by several researchers I would think that FCS also contains albumin and other proteins/chemicals that may modify the response of the drug. Albumin binds many drugs.