IGF1 stimulation of THP1 cells - (Sep/28/2012 )
We plan to investigate the miRNA expression profile in THP1 cells after IGF1 stimulation. The aim is to identify biomarkers related to IGF1-doping in professional sports.
Is there anybody, who is experienced in stimulating cells and can recommend any good IGF1-concentration in cell culture? We have chosen to use amounts which are in the region between ED50 until 10-times higher (10ng/ml and 125ng/ml). But there is also literature describing that more than 750ng/ml was used.
In addition, does anybody know good markers for best IGF1 stimulation? So far, we are using CSF1, VEGF, and PHLDA (which are all described to be IGF1 responsive).
I would recommend you produce a dose response curve for this type of work, and determine your own EC50 in your hands and in your specific cell type. This should be easy as you have a dose range I would do 7 doses, it will be essential to the the top and bottom of your sigmoid curve.
This might seem a bit repetitive of published work but in my experience is essential (particularly if you want a good publication) as peptide ligands can be very tricky to work with as they can "stick" to plastics tips etc and each lab uses different consumables so variations are common.
In terms of read out are you looking for a secreted marker? But if you are looking for receptor activation I have done work with a phospho-IGf1R antibody: http://www.cellsignal.com/products/6113.html which is quite good.
This will give you a absolute measure of activity as this is the first signalling event in the IGF-1 cascade.
Hope this is usefull