Dear Wanessa,

In case you haven't tried this already; I have found that the best thing to do with both the Dunn and the Zigmond chambers is to first find out whether or not you are actually generating a gradient. You can do this with a drop of food dye in one of the rings and water in the other. You may find that the fluids are being mixed at the outset of the assay thereby generating chemokinetic movements rather than chemotactic ones. Sometimes the assay gradient is easier to set up with the attractant in the centre, sometimes easier when it is placed in the outer ring, it can be a bit hit and miss that way. Unfortunately food dye tends to produce a cringe reaction in the cells otherwise I would recommend using it during the real folate assay as a check. It can be a bit fiddly but it really does work, good luck!

Natalie Andrew

On 13 Jun 2012, at 10:55, Wanessa Cristina DE LIMA wrote:

> Dear all,
> 
> I’ve been trying to set up a protocol for folate chemotaxis using a Dunn chamber (DCC100, Hawksley), but without success. I tried to build a gradient (in phosphate buffer) using 0.1 to 10mM folate, observing from 30 min to 6 hours. Although my cells DO move a lot (even after 6 hs in the buffer), they don’t do it in an oriented manner. Does anyone have any idea why this is not working, what I'm doing wrongly? I thought this was a pretty straightforward protocol… 
> 
> Any suggestions/tips are more than welcomed!
> 
> Cheers, Wanessa
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