Optimization of culture conditions for long-term primary neurons culture in 96-well microplate
To determine appropriate culture conditions for high-throughput imaging of endogenous fluorescent synapses in live primary neurons, we performed a systematic comparison and optimization of neuronal culture procedures, reagents, and media using our HCS system.
We first compared several types of 96-well microplates that were potentially suitable for HCS. Thick bottom plates precluded high quality imaging; thin bottom plates were required for imaging small and relatively weakly endogenous fluorescent synaptic structures.
We compared several types of coating reagent including gelatin, Polyethylenimine (PEI), and poly-d-lysine (PDL). Plastic (P96-1.5 P, Cellvis) and glass (P96-1.5H-N, Cellvis) bottom microplates were coated with 50 µL of 1% gelatin, 0.02% PEI, or 100 µg/mL PDL…
