Hello all,

I am using sep (super ecliptic phluorin) tagged AMPAR subunits to monitor receptor trafficking. Previously, sep-tagged GluA1 and GluA2 entered spines, giving nice punctate signals, and I could directly see exocytosis events as receptors were delivered to the plasma membrane (it's really cool!) Then one day, the exocytosis event frequency plummeted, the spine punctate signals were lost, and the green signal looks somewhat aggregated. This is the problem I'm trying to solve. The system is dissociated hippocampal cell cultures, and I'm using lipofectamine to transfect the neurons. The neurons themselves still look healthy (nice spines, spontaneously active). I have tried a lot of things to try and solve this, including;

Changing all media/glassware

Different stocks of cell culture reagents/lipofectamine

Multiple (4+) plasmids (all variants on sep-GluA1/2)

Reprepping plasmid DNA

And various other things

Any ideas? Any help would be very much appreciated. Has anyone else seen this aggregation/mislocalisation?

Thanks

Thomas