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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 "Reconstitu.......

Representative 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.......

Acknowledgements

We thank Aimee Rogers for providing hands-on training and continued product support. We thank Dr. Amanda Burke for ongoing troubleshooting and clarifying discussions. We thank Meredith Joheim and the UAMS Science Communication Group for the grammatical editing and formatting of this manuscript. This study was supported by NIH R25GM083247 and NIH 1R01CA258673 (A.R.A).

....

Materials

NameCompanyCatalog NumberComments
1.5 mL Microcentrifuge TubesFisher Scientific02-682-003Basix, assorted color
15 mL Falcon TubesBecton Dickinson Labware Europe352009Polystyrene
25 mL Serological PipetsFisher Scientific14-955-235
5 mL Round Bottom Polystyrene Test TubeFalcon352052
500 mL Vacuum Filter/ Storage Bottle SystemCorning431097
70 μm cell strainerFisher Scientific08-771-2
Adult Brain Dissociation KitMiltenyi Biotec 130-107-677Contains Enzyme P, Buffer Z, Buffer Y, Enzyme A, Buffer A, Debris Removal Solution
Aluminum FoilFisher Scientific01-213-105
Anti-ACSA-2-PE-Vio770, mouse, clone REA969Miltenyi Biotec130-116-246
Anti-Myelin Basic ProteinSigma-AldrichM3821-100UG
Anti-PSA-NCAM-PE, human, mouse and rat, Clone 2-2BMiltenyi Biotec130-117-394
BD LSRFortessaBD
BSASigma-AldrichA7906-50G
CD11b-VioBlue, mouse, Clone REA592Miltenyi Biotec130-113-810
CD31 AntibodyMiltenyi Biotec130-111-541
Ceramic Hot Plate StirrerFisher Scientific11-100-100SH
Dimethyl SulfoxideFisher ScientificBP231-100
EthanolPharmco by Greenfield Global111000200
Falcon 50 mL Conical Centrifuge TubesFisher Scientific14-432-22
Fine Scissors - SharpFine Science Tools14060-09Perfusion
FlowJoBD(v10.7.0)
gentleMACS C TubesMiltenyi Biotec130-093-237
gentleMACS Octo Dissociator with HeatersMiltenyi Biotec130-096-427
Gibco DPBS (1X)ThermoFisher Scientific14190144
Glass BeakerFisher Scientific02-555-25A
Heparin sodiumFresenius Kabi504011
LIVE/DEAD Fixable Aqua Dead Cell Stain KitThermoFisherL34965
Magnetic Stir BarFisher Scientific14-513-51
Noyes Spring ScissorsFine Science Tools15012-12Dissection
ParaformaldehydeSigma-Aldrich441244-3KGPrilled, 95%
Pipette tips GP LTS 20 µL 960A/10Rainin30389270
Pipette Tips GP LTS 250 µL 960A/10Rainin30389277
Pipette tips RT LTS 1000 µL FL 768A/8Rainin30389213
Rainin Pipet-Lite XLS (2, 20, 200, 1000 μL)Rainin30386597
RBXMO FITC XADSFisher ScientificA16167
Round Ice Bucket with LidFisher Scientific07-210-129
Round-Bottom Tubes with Cell Strainer CapFalcon100-0087
S1 Pipet FillersThermoFisher Scientific9541
Spatula & ProbeFine Science Tools10090-13Dissection & Perfusion
Surflo Winged Infusion Set 23 G x 3/4"TermunoSV-23BLKButterfly needle
Test Tube RackFisher Scientific14-809-37
Thermo Scientific Legend XTR CentrifugeThermoFisherdiscontinuedOr other standard table top centrifuge
Variable-Flow Peristaltic PumpFisher Scientific13-876-2Low-flow model
VetFlo Starter Kit for MiceKent ScientificVetFlo-MSEKITAnesthesia mask, tubing, induction chamber, charcoal canisters
VetFlo Vaporizer Single Channel Anesthesia SystemKent ScientificVetFlo-1210S0.2–4 LPM
Vi-CELL XR Cell Viability AnalyzerBeckman Coulter Life Sciences731196Cell Counting
Vi-CELL XR 4 Bags of Sample VialsBeckman Coulter Life Sciences383721Cell Counting

References

  1. Andersen, P., Morris, R., Amaral, D., Bliss, T., O'Keefe, J. . The Hippocampus Book. , (2007).
  2. Kempermann, G., Kuhn, H. G., Gage, F. H. Genetic influence on neurogenesis in the dentate gyrus of adult mice. Proceedings of the National Academy of Sciences o....

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