I want to check the effect of inhibitor on the gene expression after 24 hours. I grew the cells in serum free media for 24 hours then I give the treatment with the inhibitor and activator of the pathway. I have two questions:
1) Activator or inhibitors are to be added to the cells in the presence of FBS or absence?
2) Both activator and inhibitor should be incubated together for 24 hours or once treatment is given for specific time and then replace the cells with complete medium?
if you are looking for signaling or gene expression, it is better without serum because serum may contain some of the same inhibitors and activators that might affect the dose you are using and end up with different results.
1. absence of FBS
2. after treatment with inhibitor/activator you don't need to put the cells back in complete media....(avoid serum while using any kind of treatments as various unidentified serum factors mess up with your treatments)
good luck
Anyway: you should compare the different conditions "before" and "after" the activator or inhibitor administration. The only difference should be this, otherwise you're not in a controlled situation. Whatever medium you adopt during starvation, you should try to maintain.
Hi Deeksha,
I agree with all the above responses regarding serum. I am not sure what your experiment is but I would do each separate condition and also combinations.
Dear Ms Deeksha,
as mentioned above you should maintain the medium and you should not use the serum. You are comparing activator and inhibitor for gene expression so it is important that you should perform all the experiments simultaneously with identical condition and compare with control where you should not add any activator or inhibitor. I will suggest you to include a combination of both activator and inhibitor and a vehicle control where you should only add the solution in which you have prepared activator or inhibitor.
best of luck
Dear Deeksha,
Its always better to give treatment in serum free media. Serum is a rich medium and it contains many componenet which can inhibit or activate your gene of interest.So you never know exactly how and upto what extent your test compound is affecting the gene expression.
.I am looking for MAPkinase signalling pathway(inhibiting ERK1/2)..
If I have to check the effect on cell proliferation also along with gene expression Then do I need to add FBS. because in the absence of serum free media cell will not proliferate at all..How will I check that whether the activator or inhibitor actually effecting the cellular proliferation...
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