Obtaining Various Neural Cell Populations from a Rat Hippocampus

Abstract

Source: Schwarz, J. M. Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue. J. Vis. Exp. (2015)

This video demonstrates a protocol to isolate cells from rat hippocampi, including neurons, astrocytes, and microglia.

Protocol

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

1. Tissue Collection

1. Prepare Buffer X from the Neural Dissociation Kit by adding beta-mercaptoethanol to Buffer X to a final concentration of 0.067 mM (for 4 samples, add 13.5 µl of 50 mM beta-mercaptoethanol to 10 ml of Buffer X).
2. Prepare Enzyme Mix 1 by mixing 50 µl of Enzyme P with 1,900 µl of Buffer X for each sample as described in the protocol for the Neural Dissociation Kit For Example, for 4 samples, mix 200 µl of Enzyme P with 7,600 µl of B....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Neural Dissociation Kit (P)	Miltenyi Biotec	130-092-628
Myelin Removal Beads II	Miltenyi Biotec	130-096-733
LS Columns	Miltenyi Biotec	130-042-401
QuadroMACS Separator	Miltenyi Biotec	130-090-976
MACS MultiStand	Miltenyi Biotec	130-042-303
Nylon Mesh Sheet	Amazon	CMN-0074-10YD	40 inch width, 80 micron size mesh
Fc Block / anti-CD32	BD Biosciences	BDB550270	Reactivity for rat
APC-conjugated CD11b antibody	Biolegend	201809	Reactivity for rat
Rabbit anti-GLT1	Novus Biologicals	NBP1-20136	Reactivity for rat or human
PE-conjugated anti-rabbit secondary antibody	eBioscience	1037259	Secondary antibody for anti-GLT1
FITC-conjugated anti-rat CD90 (Thy1) mouse antibody	Biolegend	202504	Reactivity for rat