Adult brain workflow - 
Isolation and analysis of neural cells

  • Full workflow for isolation and analysis of neural cells from adult rodent brain (older than P7)
  • Generate highly purified neural cells from the adult brain in just half a day
  • Compatible with many downstream applications, such as single-cell sequencing or flow cytometric analysis

Adult brain workflow steps

Overview

Our adult brain workflow offers a fast, easy solution to isolate and analyze neural cells from adult rodent brain (>P7). Using the Adult Brain Dissociation Kit, mouse and rat with our gentleMACS™ Octo Dissociator with Heaters, gently dissociates brain tissue obtaining highly pure and viable neural cells.  Your neural cells of interest can then be isolated with our MicroBeads. These isolated cells are ready for downstream applications, including cell cultivation or analysis with optimized neural media and specific antibodies.

 

Tissue dissociation and sample clearing

Dissociation of adult brain tissue is fast and easy with our dissociation kits that are made especially for mice and rats older than P7. Together with the gentleMACS Octo Dissociator with Heaters, neural cells are processed gently and efficiently. The additional debris removal and erythrocyte lysis steps of the Adult Brain Dissociation Kit help to purify the neural cell population. For cancer research, we offer the Brain Tumor Dissociation Kit, which is particularly suited for tumor tissue. 

 

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Highly purified cells after sample clearing

The cell suspension from adult brain tissue contains a significant amount of cell debris (A) and erythrocytes (B), which hampers subsequent cell isolation, cultivation, and analysis. The Adult Brain Dissociation Kit optimizes the tissue dissociation process, and after sample clearing yields highly purified, living cells with much less cell debris (C) and erythrocytes (D).

Cell separation

To isolate specific neural cell types, we offer a wide range of MicroBeads for magnetic isolation of astrocytes, microglia, oligodendrocytes, and neurons. For neurons, we developed the Adult Neuron Isolation Kit. Through depletion of all non-neuronal cells, neurons remain untouched and thereby undergo a very gentle cell separation, which results in a high viability and purity.

 

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Isolation of adult astrocytes

A single-cell suspension from a 3 month old mouse brain was prepared using the Adult Brain Dissociation Kit, mouse and rat. Adult astrocytes were isolated from the single-cell suspension with the Anti-ACSA-2 MicroBead Kit, mouse. Cells were fluorescently stained with the Anti-ACSA-2 antibody and analyzed by flow cytometry on the MACSQuant Analyzer.

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Isolation of adult neurons

A single-cell suspension from a 3 month old mouse brain was prepared using the Adult Brain Dissociation Kit, mouse and rat. Subsequently, adult neurons were isolated from the single-cell suspension with the Neuron Isolation Kit, mouse. Cells were fluorescently stained with antibodies specific for non-neuronal cell marker and the ACSA-2 antigen and analyzed by flow cytometry using a MACSQuant Analyzer.  
 

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Isolation of adult microglia

Isolation of mouse microglia using the CD11b MicroBeads, human and mouse from a 2 month old mouse brain. The cell separation using the MicroBeads resulted in a high purity of 96,5% and a viability of 95,2 %. Cell were stained with CD11b-FITC and CD45-APC for further analysis. 

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Isolation of adult pre-oligodendrocytes

Single-cell suspension of adult brain tissue was used for cell separation with anti-O4 MicroBeads, human, mouse, rat. Cells were stained with Anti-O4-PE to verify a pure cell population of pre-oligodendroytes. 

Downstream applications

Cells processed with our adult brain workflow are compatible with many downstream applications:  

  • Cells can be stained and imaged with a variety of pure antibodies against many neural cell types.
  • Isolated cell populations are ready for further cultivation using our MACS Neuro Medium together with the MACS Neurobrew-21 or the AstroMACS medium for astrocytes.
  • Tissue dissociation with our Adult Brain Dissociation Kit, mouse and rat is ideal for analysing isolated neural cell populations via single-cell sequencing.
  • With the Adult Neural Stem Cell Sorting and Analysis Kit, mouse, neural stem cells from the subventricular zone can be cultivated or analyzed.

Cultivation and staining of adult neural cells

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Primary mouse neuron cultivation

Primary adult neurons were cultured in MACS Neuro Medium and MACS Neurobrew-21 for 7 days after isolation from adult brain tissue. Cells were fixed and stained with anti-MAP2 (green) and anti-βIII Tubulin (red) antibodies. 

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AstroMACS separation buffer and medium enhances growth of astrocytes

Adult astrocytes were isolated from 8-week old mouse brains using the Adult Brain Dissociation Kit and the Anti-ACSA-2 MicroBeads. When using the AstroMACS Separation Buffer and the AstroMACS Medium, growth of astrocytes is strongly improved (A) in comparison to astrocyte culture without the AstroMACS Separation Buffer (B). Cells were fixed and stained with GLAST Antibody (green) and DAPI (blue).

Single-cell sequencing of adult neural cells

Using our adult brain workflow, your neural cells are gently isolated for downstream applications, including single-cell analysis.

 

Single-cell analysis of both cell populations showed the main glial subtypes in the unseparated neural cell population and several neuronal subpopulations in the isolated neurons fraction. Single-cell analysis of unseparated neural cells and isolated neurons:

 

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Single-cell analysis of unseparated neural cells

Single-cell analysis of non-sorted adult neural cells led to identification of 24 different subclusters, representing neuronal and different glial cell types, including astrocytes, microglia, oligodendrocytes, vascular cells, and immune cells.

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Single-cell analysis of isolated neurons

Isolation of neurons using the Neuron Isolation Kit resulted in an increased resolution of neuronal clusters, including several subpopulations of immature and mature neurons. Moreover, the amount of non-neuronal cell types was significantly decreased. 

Cultivation and differentiation of adult neural stem cells 

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Cultivation and differentiation of adult neural stem cells

Adult neural stem cell sorting and analysis kit, mouse results in a pure population of neural stem cells from the subventricular zone, which is ready for culturing of neurospheres (A, B) and differentiation into neuronal and glial cells (C-F).

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