Genetic expression suppressor screen
Alexandros C Kokotos, Tim Ryan
Abstract
This protocol describes a low-throughput genetic screen to identify glycolytic enzymes that improve neuronal function under hypometabolic conditions.
Before start
Steps
Neuronal culture and transfection
Use cultured rodent primary hippocampal neurons, transfected with vGlutI-pH, mTagBFP2 and a construct expressing a single glycolytic enzyme, as explained in dx.doi.org/10.17504/protocols.io.ewov1qxr2gr2/v1.
Live-cell imaging
Remove the cells from the incubator and bathe them in Tyrodes containing 0.1 mM glucose.
Mount the coverslips with the cells on the imaging chamber and image as explained in dx.doi.org/10.17504/protocols.io.q26g7pn4qgwz/v1.
Locate cells in an unbiased manner using the mTagBPF2 channel and center on their axon, judging by their morphology.
After a total of 0h 5m 0s that the cells have been in the 0.1 mM glucose tyrode buffer, start the live-cell imaging experiment in the vGlutI-pH channel.
Stimulate the cells with 100 APs delivered at 10 Hz every 0h 1m 0s, for a total of 10 rounds of activity.
At the end of the experiment, perfuse cells with Tyrodes containing 50 mM NH4Cl.
Analyze the collected data accordingly.
