Time-Lapse Imaging of Mouse Neural Stem Cell Division Using Confocal Microscopy

Samenvatting

Source: Daynac, M., et. al. Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics. J. Vis. Exp. (2015)

This video demonstrates time-lapse imaging of neural stem cells (NSCs) from a transgenic mouse brain using a confocal laser scanning microscope. NSCs fluoresce red during the G1 phase and are non-fluorescent in other cell-cycle phases. An adhered NSC culture is placed in the imaging chamber, imaging parameters are set, and images are captured at regular intervals for the desired duration.

Protocol

1. Basic Setup Prior to Culture and Videomicroscopy

1. Use glass-bottom culture plates or µ-Plates for confocal video microscopy. For 1 - 5 x 10³ cells/well, use 96-well plates, and for more than 5 x 10³ cells/well, use 24-well plates.
2. At least one day prior to commencing the experiment, prepare sterile Poly-D-lysine (PDL) coated plates for adherent monolayer cultures. Add enough PDL (10 µg/ml in distilled water or dH₂O) to coat the bottom of each well and incubate overnight (O/N) at 37 °C. Remove the PDL solution and rinse three times with dH₂O before allowing the plate to dry in the hood during a....

Resultaten

Figure 1: Live analyses of cell cycle using FUCCI-Red mice. (A) Schematic representation of FU.......

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
96-well uncoated glass bottom culture plates	MatTek Corp.	P96G-1.5-5-F
µ-Plates 96-well	Ibidi	89626
Poly-D-Lysine	Millipore	A003E
NeuroCult NSC Basal Medium	STEMCELL Technologies	5700
NeuroCult NSC Proliferation Supplement	STEMCELL Technologies	5701
Heparin	STEMCELL Technologies	7980
EGF	Millipore	GF144
FGF-2	Millipore	GF003
Papain	Worthington	LS003119
EBSS	Invitrogen	24010-043
L-cysteine	Sigma	C-7352
0.5M EDTA, pH 8.0	Promega	V4231
DNase I	Sigma	D5025-15KU
Trypsin inhibitor type II	Sigma	T9128	Ovomucoid
DMEM:F12 medium	Life Technologies	31330-038
20 µm filter	BD Medimachine Filcon	340622
Eppendorf microtubes 3810X	Sigma	Z606340-1000EA
BSA	Sigma	A1595	Bovine serum albumin solution
CD24 phycoerythrin-cyanine7 conjugate [PC7]	Life Technologies	A14776	Mouse IgM; clone MMA. 1:50
CD15/LeX fluorescein isothiocyanate [FITC] - conjugated	BD Biosciences	332778	Rat IgG2b; clone M1/69. 1:50
Mouse anti-human LeX antibody	BD Pharmingen	559045	Mouse IgM; clone MAM. 1:50
Alexa647 - conjugated EGF ligand	Life Technologies	E35351	1:200; Ax647-conjugated EGF ligand
CompBeads	BD Biosciences	644204
MACS LS separation columns	Miltenyi Biotec	130-042-401	Separation columns
Anti-mouse IgM microbeads	Miltenyi Biotec	130-047-301
Hoechst 33258	Sigma	861405
INFLUX cell sorter	BD Biosciences		FACS sorter
Nikon A1R confocal laser scanning microscope system attached to an inverted ECLIPSE Ti	Nikon Corp.		Confocal laser scanning microscope
NIS-Elements AR.4.13.01 64-bit software	Nikon Corp.		NIS-Elements software
Plan Apo VC 20x DIC objective (NA: 0.75)	Nikon Corp.
ECLIPSE Ti thermostated chamber	Nikon Corp.		Thermostated chamber
ImageJ	RBS
FlowJo	Tree Star, Ashland, OR
FUCCI mice	RIKEN BioResource Center, JAPAN