Obtaining a Single-Cell Suspension from Frozen Rat Brain Tissue

Samenvatting

Source: Rubio, F. J., et. al. Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue. J. Vis. Exp. (2016)

This video demonstrates the dissociation of frozen rat brain tissue into a single-cell suspension using enzymatic digestion and mechanical trituration. The prepared cells are fixed and permeabilized with ethanol for downstream applications like flow cytometry or gene expression analysis.

Protocol

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

1. Preparation Before Tissue Collection

1. Set the centrifuge to 4 ^oC.
2. Fire polish a set of three glass Pasteur pipettes with decreasing diameters of approximately 1.3, 0.8, and 0.4 mm for each sample.
3. Prepare labeled 1.7 ml tubes containing 1 ml cold Buffer A and keep the tubes on ice.
4. Prepare an ice tray containing the brain slicing matrix, spatulas and glass plates (or inverted glass Petri dish) on which to perform the dissection. Pre-....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Brain matrix	CellPoint Scientific	69-2160-1	To obtain coronal brain slices
Hibernate A low fluorescence	Brain Bits	HA-lf	Buffer A' in the protocol is used for processing tissue and cells from the dissociation to fixation steps
Accutase	Millipore	SCR005	Enzyme solution' in the protocol is used for enzymatic digestion of tissue prior to trituration
Cell Strainer, 40 µm	BD Falcon	352340	To filter cell suspension
Cell Strainer, 100 µm	BD Falcon	352360	To filter cell suspension
Falcon 5 ml round-bottom polystyrene test tube with cell strainer snap cap	BD Bioscience	352235	To filter cell suspension before passing though the flow cytometer
Pasteur Pipet, Glass	NIH supply	6640-00-782-6008	To do tissue trituration