4 replies to this topic

[Elioelio]

I've been carrying out cell proliferation assays for a while now, and my treatments have been reducing proliferation of my cells in 6 assays. However, in the 7th they have had no effect on proliferation. (Its a timed assay, so 4 separate 96 well plates at 4 time points, using WST reagent - similar to an MTT assay).

However, in this assay, I had to use a different FBS in my media that I usually use (at 10%), as the stock I was using ran out. Could this be a viable reason for the treatments not working? If so, what could be the reasons behind this?
I will try another assay but can't get hold of new FBS until next week, and I have people wanting reports of my results!
Thanks for any help

Edited by Elioelio, 19 October 2012 - 04:12 AM.

[drwho]

Yes FBS will make a difference for your assay.  The simple answer is that it comes from different animals you will always have variation in growth factors and various other things between these animals they may different ages the list could go on forever.

You should ideally batch test very set of serum you get in your key assays to see if it changes.

Most companies will send out a free sample for you to do these tests as long as you buy a large quantity.  Also they will keep you batches and send them out to you as required if you do not have space to store large quantities.  We tend to order 100 500ml bottles in a batch.

Hope this is helpfull

Edited by drwho, 19 October 2012 - 09:42 AM.

[madelingirly]

My personal experience:
I was using FBS from sigma and the growth rate of my cells was very high.
then I bought FBS from biowest, and my cells grow very slow.
I asked ur question in this bioform, and I got the same answer, yes FBS can change ur results.

[Elioelio]

Yeah was worried that would be the case. The only difference with the FBS is that it was dialysed FBS, but of course it was not from the same batch.
Still, this shows that my test results cannot be that robust if just a change of FBS makes a difference between the treatment working or not...
Thanks for you replies

[rhombus]

Dear Elioelio,

We batch test our serum, New Zealand (NZ) origin, especially for this reason. We want to have reproducibility/accuracy in our experimental results and serum is one of the biggest variables you can add to experimentation. I normal test 3 x batches of NZ serum and there is even great variability between these. We test for:-

Oxygen consumption rates in 2 cell lines (Jurkat and HEK cells)
Cytochrome C intracellular concentration (Jurkat and HEK cells)
Inducible nitric oxide synthase (in J774)
Sodium channel expression in transfected HEK 293 cells
Mitochondrial electron transport chain complexes (Complex 1, 2, 3 and 4)

All the above are very senstive cellular process and can be massively affected by using different batches of NZ serum.
We purchase NZ sourced serum as this gives us higher oxygen consumption and higher intracellular Ctyochrome C concentrations than other country sourced serum i.e. the cells appear to be healthier.

NZ ( £200)    Australian (£175)    North American (£140)    South American (£100)   European (£60)    UK (£30)

" You get what you pay for"

Hope this is useful

Kindest regards

Uncle Rhombus