Hi Sarah,

I would try with lower concentrations of cAMP, in 1nM to 100nM range,
since the dissociation constant between cAMP and cAR1 receptor is
about 30 nM. I'm guessing the explanation why it isn't working is that
there is too much cAMP and the receptors are saturated. Also, my
experience is that when they are cAMP-chemotaxing, Dicty looks
elongated, shaped like small worms (you can check that easily
visually). I got them aggregating after about 6 hours with a surface
density that corresponds to about 5*10^6 cells/ml (for what is worth,
I used AX4, at 21 C).

Let me know if you still need help.

--
Igor Segota
graduate student
Cornell University



On Wed, Jul 13, 2011 at 11:49 AM, Sarah Power <[log in to unmask]> wrote:
> Hi,
>
> I want to do chemotaxis experiences with dictyostelium (strain Ax2-GFP).
> I tried with the protocols on the website of DictyBase. For exemple, I
> tried 1% and 2% agar in Sörensen's buffer an I used cAMP at differents
> concentrations: 10mM, 1mM, 200uM, 100uM, 10uM, 1uM.  I did wells in a 6
> hole-plate(fill with agar) and added 15 ul of cAMP.  I put a drop of 1ul of
> dicty at 2, 4, and 6mm from the edge of the well and I waited 3,4 or 5 hours
> before imaging the cells.
> For the preparation of  the cells, I tried a 5 or 6 hours starvation period
> at 10 000 000 cells/ml than I resuspend cells at 250 000 000 cells/ml, I
> also tried an overnight starvation at 18oC and a period of three hours at
> 22oC before resuspending cells at 250 000 000 cells/ml.  Despite all my
> effort, the cells do not seem to move towards the gradient of cAMP as it is
> suppose to be.
> It's important for my further experiences to use cAMP for chemoattractant,
> therefore I do not want to use folate.
>
> Do you know anything that could help me?
>
> Thank you
>
> Sarah Power
>
>
>