Why do researchers serum starve the cells before studying the molecular event in a cell line. I am planning to look at the Insulin signalling events in skeletal muscle cell lines, should I serum starve them before stimulation with insulin?
Serum contains numerous kinds of growth factors and cytokines which contribute to the activation of the signal pathways related to survival and proliferation of the cells of interest. For instance, if you would try to investigate into the susceptibility to EGFR activation between the different types of cells or the cells under the different conditions (e.g. hypoxia v.s. normoxia), you have to use serum-free medium and EGF-containing serum-free medium. After all, serum contains several factors to influence on EGFR signal pathway other than EGF ligand, and further, you have to note that signal pathway is always cross-talked with other signal pathways by negative feedback or compensatory activation machinery (e.g. sustained EGFR activation down-regulates the expression level of EGFR itself, while it can also activate mTOR signaling).
That is why you have to perform serum starvation before adding insulin, otherwise you cannot obtain the exact data on the time/ concentration-dependent insulin/ IGF-1-mTOR signal activation. How long you have to put the skeletal muscle cell lines under serum starvation before adding insulin to serum-free medium, and how much concentration or how long you have to treat them with insulin, largely depends on the cell context, so that you have to perform trial and error-like experiments.
Revathy,
Serum starvation may be useful to eliminate the influence of insulin that will inevitably be present in the serum. If you use a sensitive assay for insulin measurement, you can determine the exact amount. An additional variability can come from differences in amounts of insulin present in different batches of serum.
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Serum starvation also brings all cells to phase of growth arrest thereby equalising all cells into the same phase of cell cycle.This enables pronounced effect following treatment with growth signals.
Revathy, it all depends on what questions you intend to address in your study.
There are often more than one way to approach most issues.
Regarding serum free medium, whether is it really 0% (without serum.i.e.FBS/FCS) or just a little of serum (for example 2%)?
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