Establishing Primary Mixed Glial Cell Cultures from a Mouse Spinal Cord

Abstract

Source: Malon, J. T., et al. Preparation of Primary Mixed Glial Cultures from Adult Mouse Spinal Cord Tissue. J. Vis. Exp. (2016).

This video demonstrates a technique for isolating and culturing primary mixed glial cells from adult mouse spinal cord tissue. The protocol involves enzymatic digestion of the tissue, followed by the removal of broken cell membranes and myelin debris to obtain a single-cell population. The cells are then cultured in media optimized for mixed glial cell growth.

Protocol

1. Preparation of Solutions in a Culture Hood under Aseptic Conditions

1. Prepare culture media: complete Dulbecco's modification of Eagle's media (cDMEM) containing DMEM (with 4.5 g/L glucose), 10% Fetal Bovine Serum (FBS), 2 mM L-glutamine, 100 IU/mL penicillin, 100 mg/mL streptomycin, 250 ng/mL Amphotericin B, and 50 µM 2-mercaptoethanol (2-ME; see 1.1.1). Mix and filter sterilize all other components and then add FBS. Store the culture media at 4 ^°C.
   1. Prepare 50 mM 2-ME/phosphate-buffered saline (PBS) stock solution: add 100 µL of concentrated 2-ME (14.3 M) into 28.6 mL of 1x PBS, and ....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's modification of Eagle's media (DMEM) with 4.5 g/L glucose	Lonza	12-709F	The cDMEM media is a standard, widely used culture media.  Individual researchers can decide where to purchase the DMEM and all other components used to make cDMEM media.
L-Glutamine (100x)	Lonza	17-605E	The L-glutamine is a standard, widely used component for various culture media. Individual researchers can decide where to purchase L-glutamine.
Antibiotic-Antimycotic Solution (100x)	Corning-Mediatech	30-004-CI	This is a combination of penicillin, streptomycin and Amphotericin formulated to contain 10,000 units/mL penicillin G, 10 mg/mL streptomycin sulfate and 25 µg/ mL amphotericin B. Individual researchers can decide where to purchase individual components.
2-mercaptoethanol (2-ME)	Sigma-Aldrich	M3148	A BioReagent, suitable for cell culture, molecular biology and electrophoresis.
Papain dissociation system	Worthington Biochemical Corporation	LK003150	Individual components of this kit can be purchased separately.
Percoll	GE Health Care	17-0891-01	Percoll is sold as sterile solution. Undiluted Percoll can be re- autoclaved if needed.
Lab-line incubator/shaker	Barstead/Lab-line	MaxQ4000	This is the incubator/shaker we have currently. Other types of shakers can be used instead.