Preparing a Single-Cell Suspension of Immune Cells from Murine Brain Tissue

Abstract

Source: DiSano, K. D., et al. Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune Cells. J. Vis. Exp. (2020).

The video demonstrates the preparation of a single-cell suspension of immune cells from a murine brain tissue. It involves enzymatic digestion to release cells from the brain tissue, followed by density gradient centrifugation to isolate the immune cells based on their densities to obtain the single-cell suspension.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Preparing single-cell suspensions of brain and spinal cord tissue

1. Transfer the brain or spinal cord tissue with media to the top of the 100 mm Petri dish by pouring the tissue and media from the tube. Using forceps, move the tissue to the bottom of the Petri dish. Finely mince the brain or spinal cord with a sterile razor blade. Using the razor blade, move the minced tissue to the bottom of the plate by scraping to gather the tissue.
   NOTE: For the enzymatic diges....

Results

Figure 1: Identification of CD45hi infiltrating immune cells in CNS tissues. (A) Gating strategy for the identification of CD45hi infiltrating immune cells (P1), CD45lo microglia (P2), and CD45- oligodendrocytes, astrocytes, and neurons among total liv.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine Serum Albumin	ThermoFisher Scientific	37002D
Centrifuge	Beckman Coulter	Allegra X-12R centrifuge
Collagenase I	Worthington	LS004196
Conical tube, 15 mL	VWR	525-1069
Conical tube, 50 mL	VWR	89039-658
Cover glass	Hauser Scientific	5000
Curved forceps	Fine Science Tools	11003-14
Disposable polystyrene tube, 14 mL	Fisher Scientific	14-959-1B
DNase I	Worthington	LS002139
Dry ice	Airgas	N/A
Durmont #7Forceps	Fine Science Tools	11271-30
EDTA disodium salt dihydrate	Amresco	0105-500g
Ethanol, 100%	any	N/A
Filter top tube, 5 mL	VWR	352235
Fixable viability stain 780	Becton Dickinson	565388
Glucose	Fisher Chemical	D16-500
Hank's Balnced Salt Solution (HBSS)	Corning	21-020-CV
Hemacytometer	Andwin Scientific	02-671-51B
Hemostat	Fine Science Tools	13004-14
HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid)	ThermoFisher Scientific	15630080
KCl	Fisher chemical	BP366-500
KH2PO4 (anhydrous)	Sigma Aldrich	P5655-100G
Liquid Nitrogen	Airgas	N/A
Mouse FC block (CD16/32)	Becton Dickinson	553141
Na2HPO4 (anhydrous)	Fisher Chemical	S374-500
NaCl	Fisher chemical	S671-500
Needle, 25 gauge	Becton Dickinson	305122
Normal mouse serum	ThermoFisher Scientific	31881
Nylon mesh strainer	VWR	352350
OCT	Sakura	4583
Paraformaldehyde, 20%	Electron Microscopy Sciences	15713-S	Diluted to 4% using 1 x PBS
Pasteur pipette, 9 inch, unplugged	Fisher Scientific	13-678-20C
PBS (1x)	Corning	21-040-CV
PE Rat Anti-Mouse CD4	Becton Dickinson	553730
PE-CF594 Rat Anti-Mouse CD19	Becton Dickinson	562329
Percoll density gradient media	GE healthcare	17-0891-01
PerCP-Cy5.5 Rat Anti-Mouse CD45	Becton Dickinson	550994
Petri dish, 100 mm	VWR	353003
pH meter	Fisher Scientific	13-636-AB150
Pipet-Aid	Drummond Scientific Corporation	4-000-101
Pipette 200 µl	Gilson	FA10005M
Pipette tips, 1 mL	USA Scientific	1111-2831
Pipette tips, 200 µl	USA Scientific	1111-1816
Pipette, 1 mL	Gilson	FA10006M
Prolong Diamond mountant with DAPI	ThermoFisher Scientific	P36962
Purified Rat Anti-Mouse CD16/CD32	Becton Dickinson	553141
Rabbit anti-mouse CD3 (SP7 clone)	Abcam	ab16669
Rabbit anti-mouse laminin	Abcam	ab11575
Rat anti-mouse ERT-R7	Abcam	ab51824
RPMI 1640	Corning	10-040-CV
Serological pipet, 1 mL	VWR	357521
Serological pipet, 10 mL	VWR	357551
Serological pipet, 5 mL	VWR	357543
Sodium hydroxide	Fisher Scientific	S318-100
Sucrose	Fisher chemical	S5-500
Surgical scissors	Fine Science Tools	14001-16
Surgical scissors, extra fine	Roboz	RS-5882
Syringe, 10 mL	Becton Dickinson	302995
Syringe, 5 mL	Becton Dickinson	309646
Trypan blue	Gibco	15250-061