The P19 mouse embryonic carcinoma cell line (P19 cell line) is widely used for studying the molecular mechanism of neurogenesis with great simplification compared to in vivo analysis. Here, we present a protocol for retinoic acid-induced neurogenesis in the P19 cell line.
P19 cell line is known to differentiate into neurons. However, protocol for neuron differentiation of P19 cell line are sometimes complicated. This method allow us to perform the neurogenic experiment in a user-friendly manner and expand the possibility of the usage of P19 cell line for the future.
To begin, maintain P19 cells in maintenance medium, comprised of DMEM, with 4.5 grams per liter of glucose, supplemented with 10%fetal bovine serum, 100 units per milliliter penicillin, and 100 units per milliliter streptomycin. Incubate the cell culture in a 37 degree Celsius and 5%carbon dioxide environment until the cells are approximately 80%confluent. Remove the spent medium from the cell culture flask and wash the cells with two milliliters of calcium and magnesium-free PBS.
Add one milliliter of 0.25%trypsin-EDTA to completely cover the monolayer of the cells and incubate at 37 degrees Celsius and 5%carbon dioxide for two to three minutes. Under a microscope, check to see if all the cells are detached. Resuspend the cells in nine milliliters of maintenance medium in order to neutralize the trypsin.
Transfer the cells into a 15-milliliter tube and centrifuge at 200 times G and room temperature for five minutes to pellet the cells. Then, aspirate the supernatant without disturbing the pellet. Resuspend the pellet in 10 milliliters of fresh maintenance medium and use a cell counter to determine the cell number according to the manufacturer's instructions.
Seed 20, 000 cells per square centimeter in a new T-25 flask and add up to 10 milliliters of maintenance medium. Incubate at 37 degrees Celsius and 5%carbon dioxide for two to three days. To begin trypsin digestion, first slowly aspirate the supernatant and wash the cells with two milliliters of calcium and magnesium-free PBS.
Then, add one milliliter of 0.25%trypsin EDTA to the cells and incubate them at 37 degrees Celsius and 5%carbon dioxide for two to three minutes. After the incubation, to neutralize the trypsin, resuspend the cells in nine milliliters of differentiation medium, comprised of DMEM with high glucose level and supplemented with 5%fetal bovine serum, 100 units per milliliter penicillin, and 100 units per milliliter streptomycin. Transfer the cells into a 15-milliliter tube and centrifuge at 200 times G and room temperature for five minutes to pellet the cells.
Then, slowly aspirate the supernatant and resuspend the pellet in one milliliter of fresh differentiation medium without RA.Use a cell counter to determine the cell number according to the manufacturer's instructions. For aggregate generation, start by adding 10 milliliters of differentiation medium, supplemented with five microliters of RA to a 100-milliliter, non-treated culture dish. Seed one million cells in the culture dish and incubate at 37 degrees Celsius and 5%carbon dioxide for two days in order to promote aggregate formation.
After the incubation, with a 10-milliliter pipette, aspirate the medium containing the aggregates and transfer it to a 15-milliliter tube at room temperature. Wait for 1.5 minutes for the aggregates to settle at the bottom and then discard the supernatant. Add 10 milliliters of fresh differentiation medium supplemented with 0.5 micromolar RA.Gently seed the aggregates in a new 100-millimeter, non-treated culture dish and incubate at 37 degrees Celsius and 5%carbon dioxide for two days.
To begin, use a 10-milliliter pipette to transfer the cell aggregates to a 15-milliliter tube. Wait for 1.5 minutes at room temperature for the aggregates to settle at the bottom and then discard the supernatant. Wash the aggregates with serum and antibiotic-free DMEM.
Wait for cell aggregates to settle at the bottom, discard the supernatant, and add two milliliters of 0.25%trypsin EDTA. Incubate the tube in a water bath at 37 degrees Celsius for 10 minutes, tapping every two minutes to keep the cells in suspension. Then, add four milliliters of maintenance medium in order to stop the trypsin activity and pipette up and down 20 times using a one-milliliter pipette.
Finally, centrifuge the cells at 200 times G and room temperature for five minutes. Remove the supernatant and resuspend the cell pellet in five milliliters of maintenance medium. Use a cell counter to determine the cell number and proceed with plating the cells.
To plate cells, first add three milliliters per well of maintenance medium to a six-well culture plate. Then, seed the cells at a density of 500, 000 per well. Incubate the plate at 37 degrees Celsius and 5%carbon dioxide.
Seed the cells on cover glasses in a six-well culture plate. When they reach 20%confluence, proceed to immunostaining with Anti-MAP2 antibody. Then, isolate RNA, and perform reverse transcription PCR for MAP2, NueN, OCT4, NANOG, and a GAPDH.
In this study, a simplified method for neuronal differentiation is introduced. The P19 cell line is cultured in a non-treated culture dish with 5%FBS and 0.5 micromolar RA.After four days, the cell aggregates are dissociated with trypsin and seeded on an adherent cell culture plate for the next four days. The efficiency of this method is evaluated by the comparison between RTPCR analysis of the P19 cell line in an undifferentiated state and during neurogenesis.
Results show a rapid decrease of expression of pluripotency genes, such as Oct4 and NANOG, and upregulation of neurnomal marker genes, such as MAP2 and NeuN. We believe that the method developed in this study is simple and will play a part in elucidating the molecular mechanisms of neurogenesis, as well as neurodegenerative disease.
