Isolation of Multiple Cell Types from a Mouse Brain by Mechanical Homogenization

Abstract

Source: Molina Estevez, F. J., et al. Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods. J. Vis. Exp. (2019).

This video demonstrates the isolation of multiple types of cells from mouse brain tissue. Tissue is first mechanically sheared using a tissue grinder in a salt solution to maintain an osmotic balance. The tissue homogenate is then subjected to density gradient centrifugation to remove cellular debris. Cells are then resuspended in a solution for further analysis.

Protocol

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

1. Mechanical homogenization of the brain and spinal cord

NOTE: Volumes described in this section are enough for the homogenization of one-half brain or spinal cord.

1. Pre-chill the glass mortar of the Dounce tissue grinder (Table of Materials) set on ice.
2. Add 3 mL of pre-chilled 1x Hank's balanced salt solution (HBSS) to the mortar.
3. Transfer the tissue (brain or spinal cord) from the we....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10X HBSS (Calcium, Magnesium chloride, and Magnesium Sulfate-free)	Gibco	14185-052
70 mm Cell Strainer	Corning	431751
Conical Tubes (15 mL)	CellTreat	229411
Conical Tubes (50 mL)	CellTreat	229422
Dounce Tissue Grinder set (Includes Mortar as well as Pestles A and B)	Sigma-Aldrich	D9063-1SET
Percoll	GE Healthcare	10266569	sold as not sterile reagent
Percoll	Sigma	65455529	sterile reagent (to be used for applications requiring sterility)
Percoll PLUS	Sigma	GE17-5445-01	reagent containing very low traces of endotoxin