Tracking Axonal Transport of Organelles in Motor Neurons Using a Microfluidic Device

Abstract

Source: Altman, T., et al. Axonal Transport of Organelles in Motor Neuron Cultures using Microfluidic Chambers System. J. Vis. Exp. (2020).

This video demonstrates a method for tracking axonal transport in motor neurons using a microfluidic chamber system. It involves culturing mouse embryonic spinal cord tissues in polymer-coated wells with nutrient medium to induce axonal growth. The movement of fluorescent dye-stained organelles in live imaging, confirms the bidirectional axonal transport.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Microfluidic chamber or MFC preparation

1. PDMS casting in primary molds (Figure 1)
   1. Purchase or create primary molds (wafers) following a detailed protocol.
   2. Use pressurized air to remove any type of dirt from the wafer platform before proceeding to the coating step. The surface of the wafers should look smooth and clear.
   3. Fill a container with 50 mL of liquid nitrogen. Prepare a 10 mL syringe and 23 G ne....

Results

Figure 1: Silicone mold preparation. Schematic drawing describing the procedure of chlorotrimethylsilane wafer cleansing. (A) First, 50 mL of liquid nitrogen were added to an appropriate container. Working in a chemical hood, a syringe and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
35mm Fluodish – glass bottom dish	World Precision Instruments WPI	FD35-100
50mm Fluodish – glass bottom dish	World Precision Instruments WPI	FD5040-100
Andor iXon DU-897 EMCCD camera	Andor
ARA-C (Cytosine β-D-arabinofuranoside)	Sigma-Aldrich	C1768	stock of 2mM in filtered DDW
B-27 Supplement (50X)	Thermo Fisher	17504044
BDNF	Alomone Labs	B-250	Dilute to 10 µg/mL in filtered ddw with 0.01% BSA)
Biopsy punch 1.25mm	World Precision Instruments WPI	504530	For preperation of large MFC
Biopsy punch 6mm	World Precision Instruments WPI	504533	For preperation of small MFC
Biopsy punch 7mm	World Precision Instruments WPI	504534	For preperation of large MFC
Bitplane Imaris software - version 8.4.1	Imaris
Bovine Serum Albumine (BSA)	Sigma-Aldrich	#A3311-100G	5% w/v in ddw
Chlorotrimetylsilane	Sigma-Aldrich	#386529-100ML
CNTF	Alomone Labs	C-240	Dilute to 10 µg/mL in filtered ddw with 0.01% BSA)
Density Gradient Medium - Optiprep	Sigma-Aldrich	D1556
Deoxyribonuclease I (DNAse) from bovine pancreas	Sigma-Aldrich	DN-25	stock 10mg/mL in neurobasal
Dow Corning High-vacuum silicone grease	Sigma-Aldrich	Z273554-1EA	For epoxy mold preperation
DPBS 10X	Thermo Fisher	#14200-067	dilute 1:10 in ddw
Dumont fine forceps #55 0.05 × 0.02 mm	F.S.T	1125520
Epoxy Hardener	Trias Chem S.R.L	IPE 743	For epoxy mold preperation
Epoxy Resin	Trias Chem S.R.L	RP 026UV	For epoxy mold preperation
FIJI software	ImageJ
GDNF	Alomone Labs	G-240	Dilute to 10 µg/mL in filtered ddw with 0.01% BSA)
Glutamax 100X	Thermo Fisher	#35050-038
HB9:GFP mice strain	Jackson Laboratories	5029
HBSS 10X	Thermo Fisher	#14185-045	Dilute 1:10 in ddw with addition of 1% P/S and filter
iQ software	Andor
Iris scissors, curved, 10 cm	AS Medizintechnik	11-441-10
Iris scissors, straight, 9 cm	AS Medizintechnik	11-440-09
Laminin	Sigma-Aldrich	#L-2020
Leibovitz's L-15 Medium	Thermo Fisher	11415064
LysoTracker Red	Thermo Fisher	L7528
Mitotracker Deep-Red FM	Thermo Fisher	M22426
Neurobasal medium	Thermo Fisher	21103049
Nikon Eclipse Ti micorscope	Nikon
Penicillin-Streptomycin (P/S) Solution	Biological Industries	03-031-1
Poly-L-Ornithin (PLO)	Sigma-Aldrich	#P8638	Dilute 1:1000 in flitered 1X PBS
Sylgard 184 silicone elastomer kit	DOW Corning Corporation	#3097358-1004
Vannas spring microdissection scissors, 3 mm blade	F.S.T	15000-00