I am trying to perform a proliferation assay in a pericytes culture using radiolabeled thymidine. However, the signals I receive are very low, no matter what I do. Can it be due to the 2h serum starvation and/or that the 24 h experiment is enrolled in serum free medium? Do you have any experience in using serum free medium vs serum containing medium when performing proliferation studies? Or do you have any other ideas to explain the low signals?
Hi,
I think is a good idea to include a control (non-starved cells) in your assay.
Good luck!
Regards,
Daniele
If you are looking at growth factor signaling, then you must starve your cells to see the largest effect. If your cells do not die following serum withdrawal then I would leave it out. For a positive control you can add serum to one group (assuming that a growth factor for pericytes exists in blood).
Second point, for the thymidine incorporation assay, 24 hours is 2 short. For a cell that is in G1 it will take about 24 hours to enter and complete S-Phase. If the cells are in Go, it may take longer. I would increase you Thymidine incorporation time to 48 hours. When I did this with hepatocytes, there is a huge cell density effect. High cell density lead to very low growth factor thymidine incorporation and low density lead to a large effect. This is contact inhibition.
Here is an old paper describing method.
http://www.ncbi.nlm.nih.gov/pubmed/1531940
Complete serum starvation is not good for many types of cells. The cell cycle may stop at G1 phase after 18-24 hours of serum starvation and cells will eventually die. I personally checked that when HUVEC or microvascular endothelial cells transferred from 5-10% serum containing medium to complete serum free medium around 20-30 % cells will die within 24 hours and almost 100% cell will die after 48 hours. Therefore, complete serum free medium may not be good for proliferation assay.
Thank you all for the answers!
We have decided to do a pilot experiment with and without serum in the medium in the proliferation assay in order to compare and decide which option's the best. I only hope that the serum do not interact with the peptides we are exposing the cells for.. but we will see. But thank you for all the great input!
Nina, most cells require serum for growth. And many of the cells that do grow without serum need a while to produce enough autocrine factors to condition the media before they start growing. With that in mind, it is unlikely for you to detect any growth shortly after serum starvation.
You decision to run a pilot experiment on growth with and without serum will very likely tell you the answer you are looking for.
First, compare 3H-thymidine incorporation after the serum starvation with cells receiving serum-free medium (but contains 0.2% bovine serum albumin, BSA) with normal 10% serum medium.. Then, either longer (48h) incubation or shorter (with increased specific activity) with thymidine might help to increase the signal:noise ratio. Our experience suggests supplementing "serum-free" medium with BSA is essential for any growth factor (i.e. CaSR) addition experiments.
No, in general you should not add serum. However, if cells do not grow well you may add 0.5-1% serum for tumor cells.
I have not been clear. By "serum-free" conditions I mean medium which does not contain fetal calf/bovine serum but does contain a small amount of bovine serum albumin (i.e. 0.2%). This condition is compared with cells in "normal "medium which contains fetal calf/bovine serum (5 -10%).
I think 0.5% CS Can be added. It's expected that in absence of serum transfection will be good but for growth of the cells minimum serum( 0.5 %) should be there in the cell culture medium.
Hello Nina,
To be precise, I have tried serum containing and serum starved conditions to see the effect of some cocktail on proliferation of IMR cell line and I didn't observe any significant difference in proliferation during serum containing and serum starved conditions. Your trend generally remain same.
But if your cells are dying during experiment due to serum starvation then it might affect your proliferation results and can result into false negative/positive results.
What I SUGGEST IS TO USE SERUM IN YOUR EXPERIMENT, and use a proper control containing that serum as proliferation caused by serum in any of the treatment group will be normalised by serum used in control and hence true proliferation can be deduced without any cell death due to serum starvation.
Hope it helps !
If you include serum then you will not be able to distinguish a complete mitogen from a co mitogen.
If your cells require serum then you must use it but then you can only say that "in the presence of serum, agentX stimulated a mitogenic effect in my pericytes". You now have to figure out who it plays with because it is likely a co-mitogen. Very tricky situation that will require very careful language to describe and experiments to explore with.
Hello Nina,
Proper control cells will be used with the same conc. of FBS and the final values should be normalised with that control. In that case the final proliferation is not because of FBS but due to mitogen only as we have nullified the effect of FBS running parallel control with same conc. of FBS as that of treatment group. Infact your cells can starve in absence of FBS and true effect may not be observed. And serum is present in normal conditions (even in human body, you can't exclude that). So I dont think that using serum will cause any technical problem.
Hope it helps !
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