Differentiation of a Human Neuroblastoma Cell Line into Mature Neurons
Transkript
On day zero, count the cells using a hemocytometer. Then, dilute the cell suspension to 50,000 cells per milliliter using basic growth media. Plate 2 milliliters of cells per 35-millimeter dish, for a total of 100,000 cells per dish. Subsequently, place the cells in the incubator at 37 degrees Celsius.
On day one, add RA to the warmed and equilibrated media. Gently aspirate the old media and discard. Next, add 2 milliliters of differentiation media number one with RA to each 35-millimeter dish, and return to the incubator.
On day seven, gently aspirate the old media and discard. Add 200 microliters of warmed trypsin-EDTA to each 35-millimeter dish. Then, place the cells in the incubator for two to three minutes or until they are visibly lifted from the plate, as observed under a microscope.
Quench the trypsin by adding 2 milliliters of differentiation media number one with RA to each 35-millimeter dish, and use the media to rinse the remaining neuronal cells of the plate. Then, transfer the full volume to a 50-milliliter conical tube.
Gently triturate up and down slowly with a 10-milliliter plastic pipette, no more than five times. After that, aliquot 2 milliliters of the cell suspension into the fresh 35-millimeter dishes, before returning them to the incubator.
On day ten, gently aspirate off the media and discard. Add 200 microliters of warmed trypsin to each 35-millimeter dish, and incubate at room temperature for approximately one to two minutes, or until the neurons are visibly lifted from the dish, as observed under a microscope.
Day 10 of the protocol is particularly difficult because of the sensitivity of the cells at this stage of differentiation. It is imperative to minimize the time the cells are in trypsin to approximately one minute at room temperature, and then put the cells immediately back into the incubator.
Then, quench the trypsin by adding 2 milliliters of differentiation media number two to each 35-millimeter dish, and use the media to rinse the remaining neuronal cells of the plate. Afterward, transfer the contents to a 50-milliliter conical tube. Gently triturate up and down slowly with a 10-milliliter plastic pipette no more than five times. Dispense 2 milliliters of cell suspension into ECM-coated 35-millimeter dishes before returning them to the incubator at 37 degrees Celsius.
On day 11, gently aspirate off the old media and discard. Slowly, add 2 milliliters of differentiation media number three with RA to each 35-millimeter dish before returning them to the incubator.
On day 18, change the media to fresh differentiation media number three with RA once every three days to maintain neuron health. Do not allow neurons to be exposed to air for an extended period of time.
