Hi, Rajesh!

Do you mean, that you have normal patch, but after application of agonist you lose the cell?

If it so, then there are two possible reasons: 1) May be you have too high pressure in the application system and the cell just flowing away. Try to put the tube little bit away from the cell. Try different locations of it, or decrease the pressure if possible. 2) May be you have a lot of expressed NMDA receptors in the cell -> try to decrease plasmid concentration during transfection, or as the first step - decrease the agonist concentration.

No need to use antagonists if you want to activate receptors!

I also can advise to use specific agonist for NMDA receptors and not Glutamate. Glutamate is rather "dirty compound" and has many targets.

Usually people use 100 µM NMDA + 10 µM glycine.

Glycin always should present in the agonist solution, as it is a co-activator, and NMDA receptor can't be activated without it. But I suppose you add it to glutamate.

Best,

Elina

Hi Rajesh,

I agree with Elena's advice.

Are your cells healthy? Which subunits are you expressing?

Do you have a high"leak" current when voltage clamp?

Best,

Kevin

I "hate" electrophysiology since I do not even know how to interpret the data. Also I have the impression that there are a lot of tricks to learn or it seems so (to me). In brief, having had contacts with electrophysiologists: if I were young and interested in electrophysiology I would go to a very well reputed lab to learn.

I agree with Elena's and jones answer. after how many hrs of transfection you are patching the cells. is osmolarity of solutions and pH are in range? are you adjusting the Rs compansation.

first of allyour cells shuld be healthy and properly stic on the base. you can add littile more ca for making strong seal not the Mg as it will block the NMDA. so make sure that which subunit of the NMDA u are expressng.

good luck

Thanks for all suggestions...

Is it that the cells are dying, and you are looking for a way to keep them alive? One of the problems that I had when I was working to express NMDA receptors is that the medium has glutamate in it, which opens the receptors allowing a large calcium influx which kills the cells. We got around the problem two ways. First, we grew transfected cells in an antagonist to block the receptors. We used CPP. However, keeping an appropriate amount of CPP in the medium can be expensive. Mg block might work, though I have never tried it. Also, polyamines might work. Again, I never tried those. A second way is to do transient expression in the cells, and then harvest them before they die. We used viral transduction of NMDA 2A and NMDA 2B subunits and then followed cell death. We then used the cells before there was a large amount of cell death.

Thanks James...

Are the HEK293 cells unhealthy before adding the agonist via the theta glass? If so, you should indeed add an antagonist to the cells after transfection. That is in fact what we mostly do when transfecting with GluN1 + 2A and 2B. In addition, it is not so expensive since the volume is small (e.g. 1 ml in a 24 well plate). You can also add Mg, but it will not prevent opening of NMDARs if the cell depolarizes. We usually use APV.

There are several ways to increase the stability of patches during the application of glutamate (if that is your problem). Do you pull outside-out patches? If so, you don't want to apply the solution with a very high speed (test the optimal speed with open tip pipettes and different salt-solution concentrations in the theta glass). In addition, the angle of the patch pipette should be close to perpendicular to the theta glass. If you lift whole HEK293 cells you should have the pipette parallel to the theta glas. The strong solution flow would blowing the HEK cell from the pipette if you the angle is 90 degrees (so different angles for outside-out patches and whole cells). The good thing about whole HEK293 cells is that you have more NMDA receptors and consequently currents are bigger. The bad thing is that the current kinetics are a little bit slower than on outside out patches due to the much bigger size of the entire cell (NMDA receptors on different parts of the cell "see" glutamate at different times). This is most likely not so relevant if you study slow receptors like NMDARs, but would matter if you study fast receptors like AMPA receptors. The survival of patches or cells does not depend on the particular agonist (at least in my experience). However, NMDA receptor currents are different when applying different agonists; e.g. NMDA application results in faster deactivation than application of glutamate which can be explained by the different affinities. Let me know if you need more information and good luck with the experiments.

@Rafal Franco: Electrophysiology is great!