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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Protocol

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

Materials

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)
  • (Optional) Beta-mercaptoethanol, 50 mM
  • 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
  • (Optional) gentleMACS™ Dissociator (# 130-093-235), gentleMACS Octo Dissociator (# 130-095-937), or gentleMACS Octo Dissociator with Heaters (# 130-096-427)
  • (Optional) C Tubes (#  130-093-237, # 130-096-334)
  • (Optional) MACS Neuro Medium (# 130-093-570)
  • (Optional) (Optional) MACS NeuroBrew-21 (# 130-093-566)
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.
  • (Optional) Myelin Removal Beads II, human, mouse, rat (# 130-096-433, # 130-096-733)

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

For a detailed list of antigen compatibilities and the right choice of NTDK refer to the table on  NTDK product page at www.miltenyibiotec.com.

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 Separator
  • Pre-Separation Filters, 70 μm (# 130-095-823) to remove cell clumps
  • PEB buffer: Dilute MACS BSA Stock Solution (# 130‑091‑376) 1:20 with autoMACS® Rinsing Solution (# 130‑091-222). Prepare fresh.
    ▲ Note: Do not use autoMACS Running Buffer as it contains azide!
  • CD31 antibodies, mouse (clone 390) conjugated to, e.g., PE (# 130-102-608). Learn more about our antibodies and dyes.
  • MACSQuant® Analyzer 10

For cell culture

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