we routinerly used ca-free medium with different concentrations of FCS on film of colalgen and gelatine. I believe also fibronectin. The cells, primary osteoblasts and fibroblasts, adhere to the substratum

We tried to chelate calcium from culture media using different concentrations of sodium citrate, but it proved to be unstable, so we chose to use calcium-free and supplemental media with different concentrations of calcium gluconate.

We use DMEM calcium-free Gibco cod. 21068028

Hello Hassan, you can remove the Ca++ and other low molecular weight components by dialyzing the serum against calcium free PBS. Boil some visking tubing in water then tie a knot in one end.  Add the serum then tie off and dialyze the resulting bag(s) against PBS in a beaker with a magnetic stirrer overnight at 4 degrees C.  Filter-sterilize the serum immediately.

Thank you Raymond

João Pedro Hübbe Pfeifer thanks. my problem is not with DMEm which I found a low-calcium version of it, but eith FBS

Hi Paola, my problem is that there is no supplier who can provide me a low-calcium FBS. all the products available in North America contain a calcium concentration of around 6.5 mM. Purifying FBS is not a viable option for me. The presence of FN in the culture media instead of FBS will help the adhesion of cells on solid surfaces regardless to the content of calcium in the culture media. nevertheless, using purified VN instead of FBS will still need the presence of calcium in the culture media. 

Sorry Hassan, I do not understand why you cannot used dialysed Foetal Calf Serum with calcium-free medium.

Thank you Raymond. The problem is that a dialysed FBS or FCS will still have a large concentration of calcium. According to majority of supplier they perform dialysis to remove hormones... The concentration of calcium in DMEM is below micrometer concentration which is okay for my study. the biggest concentration of calcium is in FBS  

Dear Hassan,

Frankly I do not understand your experiment. Adherent mammalian cells require divalent cations to attach to the ECM - be that vitronectin, fibronectin, collagen, laminin and even GAGs. These are the dominant species present in serum that mediate cell attachment. The major cell surface receptors that mediate these interactions are integrins, which are shape-dependent. Their shape, and thus function, is stabilised by divalent cations. If you remove the calcium from your media (recommend that you use serum-free, because you'll never dialyse away all calcium from FCS, which its chock full of molecules that bind calcium) then adhesion will be mediated by other divalent species, e.g. magnesium, manganese, etc. So what?

Don't forget that calcium is an essential element required by a multitude of intracellular biochemical processes that support cell survival. Thus if you completely remove calcium (if you could) your cells will die rather quickly. In which scenario, what are you measuring?

What is your objective?

Hi David,

A paper by Edwards et al. (1987) indicates that cells in a culture media that contains purified fibronectin you don't need a high concentration of calcium for cell to attach on a solid surface. the paper talks about a calcium magnitude of 10e-6 mol/L. but this is not the same case for vitronectin and FBS or FCS. What I am planning to do is to study the impact of low calcium concentration in FBS or purified vitronectin on cells adhesion.  

Thanks for clarification. However, I still do not understand the experiment.

How does it relate to events that take place in vivo? Alternatively, are you merely studying an artefact of in vitro culture? If you prefer, an alternative fact!

What is 'high calcium'? For leukocytes in vivo this is ~1.4 mM. For basal keratinocytes in vivo this is 1.4 mM!

Fibronectin is a DAMP, usually only encountered by cells in vivo upon injury/trauma. It is not a 'normal' ECM/adhesive ligand. So too is FCS/FBS - the liquid component from clotted blood. Cells do not encounter serum in homeostatic conditions, and only few (eg endothelium) will encounter plasma (not the same as serum).

So why might cells interact with FN under Ca-free conditions? Because RGD is not the only motif they recognise and use to bind to FN! FN is a complex molecule having many motif's capable of supporting cell attachment; both integrin-mediated and integrin-independent. In other words, cells may bind via cation-dependent and cation-independent mechanisms! So what about vitronectin? VN is smaller than FN, however exists in vivo in multiple conformations; some support cell attachment, some do not. Like FN it also forms molecular complexes, and not just with other proteins. Have you thought about glycans (sugars)? Both FN and VN (and FCS for that matter) are commonly found complexed with glycosaminoglycans, glycolipids and lipoproteins! All of these also bind cations. (...explains why you can't simply remove calcium using dialysis.)

IMHO I think that you are studying in vitro artefacts. I reiterate, what is your experimental objective?