Hi there!

I'm trying to perform an experiment to prove one specific protein interacts and activates NMDAr. For this purpose i'm trying to use Fura-2AM dye to see changes in Ca2+ levels inside the neurons and two photon microscopy. As a positive control, I used different concentration of NMDA (from 0.1 uM to 100 uM) which I applied to the medium with neurons. Surprisingly (?), i see little activity from the neurons.

I noticed that usually people use magnesium free medium while performing this Ca2+ measurements for NMDAr activity. I suppose the reason being Mg ions block the channel and Ca2+ cant go thru. I'm just curious if 1) Mg presence in the medium could completely explain almost no activity from my neurons in regards to Fura 2AM intensities and

2) what is the relevance of such an experiment when my other experiments were done in normal neuronal medium (with Mg ions)? Can I claim effects of the protein X is due to NMDAr binding and activating when I can't get any activation signal unless I use magnesium free medium?