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I am trying to express functional NMDA receptors in HEK293 line cells for single channel recording experiments.

The HEK cells are maintained in the standard way (Thomas & Smart 2005) and transfected with NR1 and NR2x subunit cDNAs and also GFP, using either lipofectamine or calcium phosphate precipitation. GFP expression suggests successful transfection, but cells exhibiting green fluorescence are uniformly swollen and dead and it's more or less impossible to obtain a successful patch. Untransfected cells seem to remain perfectly viable.

On the basis that the toxicity might be a consequence of Ca2+ influx through open NMDARs, I've tried including a cocktail of blockers in the growth medium, including AP5, kynurenic acid and Mg2+, but the transfected cells continue to die.

Can anyone suggest anything else I should be doing to keep the cells alive? Or am I just on a hiding to nothing? Other researchers seem to have managed this (eg Medina et al 1995, Vicini et al 1998) and do not seem to be doing anything substantially different, so I'm a bit at a loss.

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  • $\begingroup$ You need to block NMDARs with 200 uM AP5 or 1 mM kynurenic acid. We have also used 10 mM MgCl2. $\endgroup$
    – user22143
    Feb 25, 2016 at 21:41

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Try perhaps lowering the level of expression of your construct. HEK cells are notorious for expressing large quantities of your transfected insert. Do you have it under a CMV promoter? Try a milder one, such as an ubiquitin promoter or similar or, even better, a doxycyclin-inducible system (e.g., clontech's tet off) so you can manage the expression levels through the dox concentration/time of induction.

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  • $\begingroup$ It is indeed using a CMV promoter, so that would be something to look at. One suggestion I've seen is to co-transfect with a high ratio of an "empty" pcDNA plasmid as a competitor to reduce expression. That sounds a bit Heath Robinson, but it would be an easier test than splicing into a new vector... $\endgroup$
    – walkytalky
    Dec 19, 2011 at 17:58
  • $\begingroup$ Sure, you can give that a try. I would expect that to lower the transfection efficiency rather than the level of expression per cell, but it's easy enough to try it right away. Good luck! BTW: I had to google "Heath Robinson": nice one! $\endgroup$
    – Aleadam
    Dec 20, 2011 at 22:30

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