Generating a Low-Density Primary Neuron Culture From Frozen Embryonic Neurons

Begin with a cryovial containing cryopreserved embryonic neurons and place it into a heating block for controlled thawing.

Next, transfer the thawed neurons into a tube and dilute them with a neurobasal medium dropwise to balance the osmotic pressure and prevent cell lysis.

This generates a homogeneous suspension with uniformly dispersed neurons.

Seed the diluted neural suspension onto a poly-L-lysine-coated multi-well plate containing a neurobasal medium enriched with nutrients and antibiotics.

Incubate to allow the positively charged poly-L-lysine to interact with the neurons via electrostatic interactions, facilitating their adherence.

Supply the wells with fresh neurobasal media.

Incubate the cells in the same media under humid conditions to prevent media evaporation.

The embryonic neurons utilize the nutrients and grow by developing neuronal processes and forming primary neurons.

The neurobasal medium maintains the primary neurons that appear widely spaced, generating a low-density primary culture with neuronal connections.