JoVE Logo

Accedi

È necessario avere un abbonamento a JoVE per visualizzare questo. Accedi o inizia la tua prova gratuita.

In questo articolo

  • Overview
  • Protocollo
  • Risultati
  • Divulgazioni
  • Materiali
  • Riferimenti

Overview

The video showcased a cell culture method for differentiating neural progenitor cells into neurons. By incubating cells in a nutrient-rich medium supplemented with growth factors, the progenitor cells matured into neurons with developed axons and dendrites. A stabilizing agent ensured cell viability and protected against oxidative damage throughout the differentiation process.

Protocollo

1. Differentiation from NPCs to neurons (Phase II)

  1. Seed about 5 x 105 mouse embryonic stem cells or mESC derivatives within 2 mL of basal differentiation medium I per well onto the 0.1% gelatin-coated 6-well plates. Randomly divide the mESC derivatives into 3 groups, as Phase II protocol 1, protocol 2, and protocol 3, respectively.
  2. Place the plate into the 5% CO2 incubator at 37 °C to allow for attachment for 6 h. Wash them twice with 2 mL of PBS.
  3. Add 2 mL of basal differentiation medium I, N2B27 medium I and N2B27 medium II (Table 1), respectively, to each well of the above groups.
  4. Place the plates into the incubator and allow them to differentiate for another 10 days. Change the corresponding medium every 2 days.
  5. Check the differentiation status and record the morphological changes as mentioned in step 3.
  6. On Day 18, evaluate the generation of neurons (β-Tubulin III positive) and determine the differentiation efficiency of the 3 protocols used in step 4.

Risultati

Table 1: Details of the 3 protocols used in phase II differentiation.

Differentiation Phase II (10d)
ProtocolsMedia
Protocol 1Differentiation naturally: With basal differentiation medium I onlyBasal differentiation medium ...

Divulgazioni

No conflicts of interest declared.

Materiali

NameCompanyCatalog NumberComments
Anti-β-Tubulin III antibody produced in rabbitSigma AldrichT2200stored at -80 °C, avoid repeated freezing and thawing
B-27 Supplement (50X), serum freeGibco17504044stored at -20 °C, and protect from light
DME/F-12 1:1 (1x)HyCloneSH30023.01Bstored at 4 °C
Fetal bovine serumHyCloneSH30084.03stored at -20 °C, avoid repeated freezing and thawing
Fluorescence microscopyOlympusCKX53
GelatinGibcoCM0635Bstored at room temperature
GlutaMAX SupplementGibco35050061stored at 4 °C
Immunol Staining Primary Antibody dilution BufferBeyotimeP0103stored at 4 °C
KnockOut DMEM/F-12Gibco12660012stored at 4 °C
MEM Non-essential amino acids solutionGibco11140076stored at 4 °C
N-2 Supplement (100X)Gibco17502048stored at -20 °C and protect from light
Normal goat serumJackson005-000-121stored at -20 °C
Neurobasal MediumGibco21103049stored at 4 °C
Nonadhesive bacterial dishCorning3262
Phosphate Buffered Saline (1X)HyCloneSH30256.01Bstored at 4 °C
Penicillin/ Streptomycin SolutionHyCloneSV30010stored at 4 °C
PD0325901(Mirdametinib)SelleckS1036stored at -20 °C
Retinoic acidSigmaR2625stored at -80 °C and protect from light
Strain 129 Mouse Embryonic Stem CellsCyagenMUAES-01001Maintained in feeder-free culture system
2-MercaptoethanolGibco21985023stored at 4 °C and protect from light
4% paraformaldehydeBeyotimeP0098stored at -20 °C
6 - well plateCorning3516
60 mm cell culture dishCorning430166
15 ml centrifuge tubeNUNC339650

Riferimenti

This article has been published

Video Coming Soon

Source: Mao, X., et al. Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro. J. Vis. Exp. (2020).

JoVE Logo

Riservatezza

Condizioni di utilizzo

Politiche

Ricerca

Didattica

CHI SIAMO

Copyright © 2025 MyJoVE Corporation. Tutti i diritti riservati