Application protocol

Isolation and cultivation of endothelial cells from adult mouse brain

In this application protocol, we present a protocol to generate highly purified and viable endothelial cells from adult mouse brain tissue. Brain tissue from mice older than P7 is dissociated into single-cell suspensions using the Adult Brain Dissociation Kit and the gentleMACS™ Octo Dissociator with Heaters for mechanical dissociation during on-instrument enzyme incubation. After dissociation, the myelin and cell debris is removed using the Debris Removal Solution and is followed by subsequent removal of erythrocytes using the Red Blood Cell Removal Solution. Endothelial cells are enriched by depletion of CD45+ cells with CD45 MicroBeads followed by a positive selection using CD31 MicroBeads.

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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.

General reagent and instrument requirements

  • Dulbecco’s phopshate-buffered saline (D-PBS) with calcium magnesium, glucose, and pyruvate. Keep buffer cold (2−8 °C).
  • Phosphate-buffered saline (PBS)
  • PB buffer: Prepare a solution containing phosphate-buffered saline (PBS), pH 7.2 and 0.5% bovine serum albumin (BSA) by diluting MACS® BSA Stock Solution (# 130-091-376) 1:20 with PBS. Keep buffer cold (2−8 °C). Degas buffer before use, as air bubbles could block the column. Always use freshly prepared buffer. Do not use autoMACS® Running Buffer or MACSQuant® Running Buffer as they contain a small amount of sodium azide that could affect the results.
    ▲ Note: BSA can be replaced by other proteins such as mouse serum albumin, mouse serum, or fetal bovine serum (FBS).

For brain tissue dissociation

  • Adult Brain Dissociation Kit, mouse and rat (# 130-107-677)
  • gentleMACS™ Octo Dissociator with Heaters (# 130-096-427)
  • gentleMACS C Tubes (# 130-093-237, # 130-096-334)
  • 35 mm diameter sterile petri dish
  • Sterile scalpel
  • Sterile forceps
  • (Optional) ART® 1000 REACH™ pipet tips (Molecular BioProducts, Inc.) for removal of dissociated material from the closed C Tubes.
  • MACS SmartStrainers (70 µm) (# 130-098-462)
  • 15 mL and 50 mL tubes
  • Centrifuge with swinging bucket rotor

For cell isolation and flow cytometry analysis

  • CD45 MicroBeads, mouse (# 130-052-301)
  • CD31 MicroBeads, mouse (# 130-097-418)
  • (Optional) Pre-Separation Filters (70 µm) (# 130-095-823)
  • MACS Columns and MACS Separators: Endothelial cells can be enriched by depletion using LD Columns followed by subsequent positive selection using MS Columns. Enrichment can also be performed by using the autoMACS Pro Separator.
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
MS1x10⁷2x10⁷MiniMACS™, OctoMACS™,
VarioMACS, SuperMACS II
LD2x10⁷4x10⁷MidiMACS™, OctoMACS™, 
VarioMACS, SuperMACS II
autoMACS5x10⁷1x108autoMACS Pro
▲ Note: Column adapters are required to insert certain columns into the VarioMACS™ or SuperMACS™ II Separators. For details refer to the respective MACS Separator data sheet.
  • Fluorochrome-conjugated Labeling Check-Reagents to stain labeled cells for flow cytometry analysis, e.g., Labeling CheckReagent-APC. Learn more about our antibodies and dyes.
    ▲ Note: The use of CD31 antibodies (clone 390) is not recommended for analysis of cells that are labeled with CD31 MicroBeads.
  • (Optional) Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cells.
  • (Optional) MACSQuant Analyzer 10 (# 130-096-343)

For cell culture

  • Double-distilled water (ddH₂O)
  • Imaging Plate CG 1.5 (96 well) (# 130-098-265)
  • Fibronectin, e.g., Human Fibronectin (Fragment) (# 130-109-393) for coating of cell culture dishes
  • EBM-2 basal medium and all supplements (Lonza, EGM™-2-MV BulletKit™, CC-3202)
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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.