Application protocol

Isolation of endothelial cells from mouse neonatal brain

The application protocol was developed to isolate high yields of viable endothelial cells from mouse neonatal brain tissue. Cells can be cultured or analyzed by flow cytometry afterwards.


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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.


The following is a listing of reagents, instruments, and consumables needed for each step of this protocol. These products are for research use only.

For brain tissue dissociation

  • Neural Tissue Dissociation Kit (P) (# 130-092-628)
  • Hanks' Balanced Salt Solution (HBSS) without Ca2+ and Mg2+ (Sigma-Aldrich # 55021C)
  • HBSS  with  Ca2+ and Mg2+ (Sigma-Aldrich  # 55037C)
  • 50 mL tubes
  • MACS® SmartStrainer (70 μm) (# 130-098-462) for 50 mL tubes
  • MACSmix™ Tube Rotator (# 130-090-753) in combination with an incubation oven at 37 °C
  • gentleMACS™ Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (#  130-093-237, # 130-096-334)
  • (Optional) MACS Neuro Medium (# 130-093-570)
  • (Optional) MACS NeuroBrew®-21 (# 130-093-566)

▲ Note: Make sure the antigen epitope that is necessary for downstream applications is preserved during the dissociation procedure. 

For a detailed list of antigen compatibilities and the right choice of Neural Tissue Dissociation Kit (NTDK) refer to the table on  the product page at

In case your epitope of interest is not listed please contact technical support.  You  can also perform  a staining experiment with this antibody after using different enzyme concentrations, i.e., different dilutions of Enzyme P or T (e.g., for NTDK (P) 1:5, 1:10; for NTDK (T)  1:2.5)  prior  to  isolation experiments to analyze the stability of your antibody epitope.

For cell isolation and flow cytometry analysis

  • CD45 MicroBeads, mouse (# 130-052-301)
  • CD31 MicroBeads, mouse (# 130-097-418)
  • LD Columns (# 130-042-901) and MS Columns (# 130-042-201) and suitable MACS Separators
  • Pre-Separation Filters (70 μm) (# 130-095-823) to remove cell clumps
  • PEB buffer (refer to "Things to prepare in advance")
    ▲ Note: Do not use autoMACS Running Buffer as it contains azide!
  • Fluorochrome-conjugated CD31 Antibody, anti-mouse for flow cytometric analysis. For more information about antibodies refer to
  • Flow cytometer, for example, MACSQuant Analyzer 10 (# 130-096-343

For cell culture

  • Fibronectin
  • EBM-2 basal medium and all supplements (Lonza, EGM™-2-MV BulletKit™, CC-3202)
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