To thaw the neurons on the day of seeding, prewarm the water bath to 37 degrees Celsius. Also, equilibrate the complete maintenance media to room temperature while protecting it from light. Meanwhile, remove the vial containing the commercially-obtained frozen neurons from the liquid nitrogen tank and transfer it to dry ice.
Then place the vial in the water bath at 37 degrees Celsius for two minutes to ensure complete thawing. Following which, disinfect the vial using 70%ethanol. Using a P1000 pipette, transfer the approximately 370 microliters of thawed neurons from the vial to a 50-milliliter centrifuge tube.
Rinse the empty vial with 630 microliters of complete maintenance medium. And using a pipette, dispense the medium dropwise into the 50-milliliter tube at a 45-degree angle. In a similar manner, using a P1000 pipette, add one milliliter of complete maintenance medium to the 50-milliliter centrifuge tube.
Then slowly add an additional two milliliters of complete maintenance medium into the tube. Next, mix 10 microliters of trypan blue with 10 microliters of the cell suspension in a micro tube. Then add 10 microliters of the mixture to a counting chamber slide for counting cells.
Following cell counting and transferring the neurons to a 15-milliliter tube, centrifuge at 400g for five minutes at room temperature. Subsequently, remove the supernatant. Using a P1000 pipette, carefully resuspend the neuron pellet in one milliliter of complete maintenance medium.
Finally, add the necessary volume of medium to achieve the desired concentration for seeding the neurons on the same day.
