Retinoic Acid-Induced Neurogenesis using Mouse Embryonic Carcinoma Cells

Abstract

Source: Leszczyński, P., et al.,  Neurogenesis Using P19 Embryonal Carcinoma Cells J. Vis. Exp. (2019)

This video demonstrates a simple technique for retinoic acid-induced neurogenesis in P19 mouse embryonic carcinoma cells. Retinoic acid activates transcription factors and triggers gene expression for neuronal differentiation.

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

Materials

List of materials used in this article

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