A Modified Roller Tube Method for the Long-Term Culture of Rodent Brain Slices

Abstract

Source: Fixman, B. B., et al. Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System. J. Vis. Exp. (2017).

This video demonstrates a modified roller tube method for the long-term culturing of brain slices from a rodent model. First, the brain slices are attached to coverslips. Then, flat-sided roller tubes with a hole on the flat side are taken, and the coverslips are positioned on the hole using adhesive discs. Finally, a nutrient medium and a carbon dioxide and air mixture are added to the tube, and the tubes are incubated with a rolling motion.

Protocol

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

NOTE: The protocol below describes the preparation and culture method for the long-term incubation and intermittent imaging of hippocampal slices. A single hippocampal slice is attached to a specially prepared photoetched coverslip using a plasma clot, and then the coverslips are sealed onto the flat side of a drilled-out roller tube, which is maintained in a roller incubator.

1. Preparation of Roller Tube Rack

1. Use the template shown in Figure 1A

Results

Figure 1: Preparation of roller tube rack. (A) Template for marking hole positions for drilling out the 15 cm tissue culture dish bottoms. If the figure is printed to the size of the scale bar shown, it can be cut out and used for marking the posi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bottoms from 15 cm culture dishes	VWR Scientific	25384-326
Phillips Head Machine Screws (#10-32)	Ace Hardware		2.5" long and 3/16" in diameter
Flat Washers #10	Ace Hardware
Machine Screw Nuts (#10-32)	Ace Hardware
Rubber Grommets	Ace Hardware		5/16", thick; 5/8", hole diameter; 1.125", OD
Polyethylene tubing (5/16"; OD, 3/16"; ID)	Ace Hardware		Cut to 1.8" length
Lock Washer #10	Ace Hardware
Drill Press, 5 speed	Ace Hardware
Nunclon Delta Flat-Sided Tubes	VWR	62407-076
Drill bits, 3 mm, 6 mm and 15 mm	Ace Hardware	Diablo freud brand	Drill bits for cutting plastic
Drill bits for wood, 1.5 cm and 1 mm	Ace Hardware
Wood file, 1/4" round	Ace Hardware
Spring clips, 16 mm snap holder	Ace Hardware
Swivel Head Deburring Tool, 5"	Ace Hardware	26307
Adhesive Silicone Sheet (Secure Seal)	Grace Bio-Labs	666581	0.5 mm Thickness
6 mm hole punch	Office Max
12 mm hole punch	thepunchbunch.com
70% Ethanol
Phototeched Coverslips, 12 mm diameter	Bellco Glass, Inc.
Bunsen Burner
Absolute Ethanol
Nanopure Water
3-aminopropyltriethoxylane	Sigma-Aldrich	A3648
Acetone	Sigma-Aldrich	179124
#5 Dumont Forceps	Fine Science Tools	11251-30
McIlwain Tissue Chopper	Ted Pella, Inc.	10180
Double Edge Razor Blades	Ted Pella, Inc.	121-6
Whatman Filter Paper	VWR	28450-182	Cut into 5.8 cm diameter circles
Poly-chloro-trifluoro-ethylene (Aclar)	Ted Pella, Inc.	10501-10	Cut into 5.8 cm diameter circles
#21 Surgical Blade	VWR Scientific	25860-144
#5 Dumont Forceps	Fine Science Tools	11251-30
Spatula, stainless with tapered end	VWR	82027-518
Gey's Balanced Salt Solution	Sigma-Aldrich	G9779
Glucose	ThermoFisher Scientific	15023-021	25% (w/v) Solution, 0.2 mm filter sterilized
Chicken Plasma	Cocalico Biologicals	30-0300-5L	Rehydrate in sterile water, centrifuge at 2500 x g 30 min at 4 °C, quick freeze aliquots in liquid nitrogen and store at -80 °C.
Thrombin, Topical (Bovine)	Pfizer	Thrombin-JMI	Quick freeze aliquots in liquid nitrogen at 1,000 international units/mL in diluent provided and store at -80°C. Use at 250 units/ mL.
Cell Roller System	Bellco Biotech	SciERA
Roller Incubator	Forma	Model 3956