NPC Neurosphere Assay: An In Vitro Assay to Assess the Migratory Response of Neural Precursor Cells from the Neurosphere

Abstract

Source: Williams, M. et al. Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs). J. Vis. Exp. (2018)

This video demonstrates the neural precursor cell neurosphere migration assay—an in vitro assay to study the migratory response of neural precursor cells from the neurosphere—via microscopic analysis.

Protocol

1. NPC Neurosphere Migration Assay

1. Neurosphere Formation
   1. Add 1 mL of 100% Expansion Media into a 35 mm dish with no coating substrate. Incubate dishes for at least 15 min at 37 °C before plating NPCs. Prepare 2–3 dishes to ensure that there will be enough neurospheres for the cell migration assay.
      NOTE: The absence of a coating substrate ensures that NPCs remain suspended in the media, which is essential for neurosphere formation. Coating dishes will prevent neurosphere formation.
   2. Lift, dissociate, and pellet cells. Resuspend cell pellet in 2–5 mL of pre-warm....

Results

Figure 1: Selecting neurospheres for migration assay. (A) Phase-contrast image of a representative field of neurospheres at 24 h. Spheres are all less than 100 μm and thus, are not collect.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Neurobasal Medium 	ThermoFischer Scientific	21103049	Component of both NIM and 100% Expansion Medium
PSC Neural Induction Medium: Protocol Link: https://goo.gl/ euub7a	 ThermoFischer Scientific  	A1647801	This is a kit that consists of Neurobasal (NB) medium and a 50x Neural Induction Supplement (NIS). The NIS is used to make 1X Neural Induction Medium and 100% Expansion Medium
Advanced DMEM/F12 Medium 	ThermoFischer Scientific  	12634-010	Component of 100% Expansion Medium
hESC-qualified Matrigel	Corning 	354277	hESC-qualified extracellular matrixmimic gel (ECM-mimic gel)
6-well plates 	Corning 	COR-3506 	Polystyrene plates used for NPC maintenance and for Neurosphere Migration Assay
Human Basic FGF-2 	Peprotech	100-18B 	Growth factor
Penicillin/Streptomycin 	ThermoFischer Scientific  	15140122	Antibiotic, component of NIM, 100% Expansion and 30% Expansion Media