Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells

Protocol

1. Aggregate Generation

1. Add 5 µL of RA (retinoic acid) (1 mM stock dissolved in 99.8% ethanol, stored at -20 °C) to the 10 mL of Differentiation Medium and mix well (final concentration of 0.5 µM RA).
   NOTE: RA is light-sensitive. A low concentration of ethanol does not affect cell differentiation.
2. Add 10 mL of Differentiation Medium (with RA) to the 100 mm non-treated culture dish (dedicated to suspension culture).
3. Seed the 1 x 10⁶ cells in the 100 mm dish (Dish surface area 56.5 cm2).
4. Put the flask with cells into the incubator at 37 °C and 5% CO₂ for 2 days to promote aggregate formation.
5. After 2 days, exchange the Differentiation Medium. Aspirate medium containing aggregates using a 10 mL pipette and transfer to a 15 mL tube at RT.
6. Allow the aggregates to settle by gravity for 1.5 min at RT.
7. Discard the supernatant.
8. Add a fresh 10 mL of Differentiation Medium with 0.5 µM RA using a 10 mL serological pipette.
   CAUTION: Do not pipette the cell aggregates up and down.
9. Seed the aggregates into a new 100 mm non-treated culture dish (dedicated to suspension culture).
10. Place the plate in the incubator (at 37 °C and 5% CO₂) for 2 days.

2. Aggregates Dissociation

1. Aspirate the cell aggregates using a 10 mL pipette.
2. Transfer the aggregates to a 15 mL tube. Allow the cell aggregates to settle by gravity for 1.5 min.
3. Remove the supernatant.
4. Wash the aggregates with DMEM alone (serum- and antibiotic-free).
5. Allow the cell aggregates to settle by gravity sedimentation for 1.5 min at RT.
6. Aspirate the supernatant and add 2 mL of trypsin-EDTA (0.25%).
7. Place the cell aggregates into a water bath (37 °C) for 10 min. Agitate the aggregates gently every 2 min by tapping with a hand.
8. Stop the trypsinization process by adding 4 mL of Maintenance Medium.
9. Pipette the aggregates up and down 20 times using a 1 mL pipette.
10. Centrifuge cells for 5 min at 200 x g and RT.
11. Remove the supernatant and resuspend the cell pellet in 5 mL of Maintenance Medium.
12. Determine the cell number with a cell counter.

3. Plating Cells

1. Add 3 mL per well of Maintenance Medium to a 6-well plate.
2. Seed cells in the 6-well culture plate at a density of 0.5 x 10⁶/well.
3. Incubate at 37 °C with 5% CO₂ concentration.

Materials

Name	Company	Catalog Number	Comments
DMEM high glucose (4.5 g/L) with L-glutamine	Lonza	BE12-604Q
Ethanol 99.8%	Chempur	CHEM*613964202
Fetal Bovine Serum (FBS)	EURx	E5050-03
Penicillin/Streptomycin 10K/10K	Lonza	DE17-602E
Phosphate Buffered Saline (PBS), 1x concentrated without calcium and magnesium ions	Lonza	BE17- 517Q
Retinoic acid	Sigma- Aldrich	R2625-50MG	Dissolved in 99.8% ethanol; store in -20 °C up to 6 months
Trypsin 0.25% - EDTA in HBSS, without calcium and magnesium ions,with Phenol Red	Biosera	LM-T1720/500
1 mL Serological Pipettes	Profilab	515.01
10 mL Serological Pipettes	Profilab	515.1
100 mm dish dedicated for suspension culture	Corning	C351029
15 mL centrifuge tubes	Sigma- Aldrich	CLS430791-500EA
5 mL Serological Pipettes	Profilab	515.05
6-well plate	Corning	CLS3516
Cell culture flasks, surface area 25 cm square	Sigma- Aldrich	CLS430639-200EA