Combined Mechanical and Enzymatic Dissociation of Mouse Brain Hippocampal Tissue

Summary

This neural cell dissociation protocol is intended for samples with a low amount of starting material and yields a highly viable single-cell suspension for downstream analysis, with optional fixation and staining steps.

Abstract

This neural dissociation protocol (an adaptation of the protocol accompanying a commercial adult brain dissociation kit) optimizes tissue processing in preparation for detailed downstream analysis such as flow cytometry or single-cell sequencing. Neural dissociation can be conducted via mechanical dissociation (such as using filters, chopping techniques, or pipette trituration), enzymatic digestion, or a combination thereof. The delicate nature of neuronal cells can complicate efforts to obtain the highly viable, true single-cell suspension with minimal cellular debris that is required for single-cell analysis. The data demonstrate that this combination of automated mechanical dissociation and enzymatic digestion consistently yields a highly viable (>90%) single-cell suspension, overcoming the aforementioned difficulties. While a few of the steps require manual dexterity, these steps lessen sample handling and potential cell loss. This manuscript details each step of the process to equip other laboratories to successfully dissociate small quantities of neural tissue in preparation for downstream analysis.

Introduction

The hippocampus was first described by a Bolognese anatomist, Giulio Cesare Aranzio, in the 1500's¹. In naming this newfound structure, Aranzio was likely inspired by its uncanny resemblance to the seahorse of the genus Hippocampus¹. The hippocampus is involved in stress responses but is widely known for its role in learning and memory. More specifically, the hippocampus is responsible for the encoding and retrieval of declarative and spatial memory¹.

The hippocampus, or hippocampus proper, is divided into the CA1 (cornu ammonis), CA2, and CA3 subfields

Protocol

Experiments were conducted in accordance with the ethical standards approved by the Institutional Animal Care and Use Committee at UAMS. 6-month-old female C57Bl6/J wild-type mice were purchased and group-housed (4 mice per cage) under a constant 12 h light/dark cycle.

1. Preparation of reagents

1. Prepare fixable live/dead stain stock solution. Reconstitute the fluorescent stain with 20 µL of dimethyl sulfoxide (DMSO).
2. Wrap the vial in foil, label it as "Reconstituted", and store it at -20 °C for up to six months.
3. Prepare a 0.9% saline solution with heparin. Dilute the contents of one vial of ....

Results

Samples were processed with a flow cytometer at a core facility, and the resulting data were evaluated with a software package for flow analysis. Previously, compensation controls were analyzed-the live/dead stain and negative control. If multiple fluorochromes are used, fluorescence minus one (FMO) controls and single-stain controls should be prepared for each antibody. Compensation for spectral overlap for the experimental samples was calculated based on the analyzed controls. For cell population identification, a hier.......

Discussion

Several steps in this neural dissociation protocol require proficient technique and dexterity–perfusion, supernatant aspiration, and myelin removal. Throughout the perfusion process, the internal organs must remain intact (aside from removing the diaphragm and clipping the heart); this includes avoiding the upper chambers of the heart with the butterfly needle. While the amount of saline with heparin needed varies, transparent fluid flowing from the heart indicates the process is complete. The brain must be complet.......

Materials

Name	Company	Catalog Number	Comments
1.5 mL Microcentrifuge Tubes	Fisher Scientific	02-682-003	Basix, assorted color
15 mL Falcon Tubes	Becton Dickinson Labware Europe	352009	Polystyrene
25 mL Serological Pipets	Fisher Scientific	14-955-235
5 mL Round Bottom Polystyrene Test Tube	Falcon	352052
500 mL Vacuum Filter/ Storage Bottle System	Corning	431097
70 μm cell strainer	Fisher Scientific	08-771-2
Adult Brain Dissociation Kit	Miltenyi Biotec	130-107-677	Contains Enzyme P, Buffer Z, Buffer Y, Enzyme A, Buffer A, Debris Removal Solution
Aluminum Foil	Fisher Scientific	01-213-105
Anti-ACSA-2-PE-Vio770, mouse, clone REA969	Miltenyi Biotec	130-116-246
Anti-Myelin Basic Protein	Sigma-Aldrich	M3821-100UG
Anti-PSA-NCAM-PE, human, mouse and rat, Clone 2-2B	Miltenyi Biotec	130-117-394
BD LSRFortessa	BD
BSA	Sigma-Aldrich	A7906-50G
CD11b-VioBlue, mouse, Clone REA592	Miltenyi Biotec	130-113-810
CD31 Antibody	Miltenyi Biotec	130-111-541
Ceramic Hot Plate Stirrer	Fisher Scientific	11-100-100SH
Dimethyl Sulfoxide	Fisher Scientific	BP231-100
Ethanol	Pharmco by Greenfield Global	111000200
Falcon 50 mL Conical Centrifuge Tubes	Fisher Scientific	14-432-22
Fine Scissors - Sharp	Fine Science Tools	14060-09	Perfusion
FlowJo	BD		(v10.7.0)
gentleMACS C Tubes	Miltenyi Biotec	130-093-237
gentleMACS Octo Dissociator with Heaters	Miltenyi Biotec	130-096-427
Gibco DPBS (1X)	ThermoFisher Scientific	14190144
Glass Beaker	Fisher Scientific	02-555-25A
Heparin sodium	Fresenius Kabi	504011
LIVE/DEAD Fixable Aqua Dead Cell Stain Kit	ThermoFisher	L34965
Magnetic Stir Bar	Fisher Scientific	14-513-51
Noyes Spring Scissors	Fine Science Tools	15012-12	Dissection
Paraformaldehyde	Sigma-Aldrich	441244-3KG	Prilled, 95%
Pipette tips GP LTS 20 µL 960A/10	Rainin	30389270
Pipette Tips GP LTS 250 µL 960A/10	Rainin	30389277
Pipette tips RT LTS 1000 µL FL 768A/8	Rainin	30389213
Rainin Pipet-Lite XLS (2, 20, 200, 1000 μL)	Rainin	30386597
RBXMO FITC XADS	Fisher Scientific	A16167
Round Ice Bucket with Lid	Fisher Scientific	07-210-129
Round-Bottom Tubes with Cell Strainer Cap	Falcon	100-0087
S1 Pipet Fillers	ThermoFisher Scientific	9541
Spatula & Probe	Fine Science Tools	10090-13	Dissection & Perfusion
Surflo Winged Infusion Set 23 G x 3/4"	Termuno	SV-23BLK	Butterfly needle
Test Tube Rack	Fisher Scientific	14-809-37
Thermo Scientific Legend XTR Centrifuge	ThermoFisher	discontinued	Or other standard table top centrifuge
Variable-Flow Peristaltic Pump	Fisher Scientific	13-876-2	Low-flow model
VetFlo Starter Kit for Mice	Kent Scientific	VetFlo-MSEKIT	Anesthesia mask, tubing, induction chamber, charcoal canisters
VetFlo Vaporizer Single Channel Anesthesia System	Kent Scientific	VetFlo-1210S	0.2–4 LPM
Vi-CELL XR Cell Viability Analyzer	Beckman Coulter Life Sciences	731196	Cell Counting
Vi-CELL XR 4 Bags of Sample Vials	Beckman Coulter Life Sciences	383721	Cell Counting