Application protocol

Isolation and cultivation of astrocytes from adult mouse brain

This application protocol describes a protocol to generate highly purified and viable astrocytes from adult mouse brain tissue. Brain tissue from mice older than P7 is dissociated into single-cell suspensions using the Adult Brain Dissociation Kit. The extracellular matrix is enzymatically digested using the kit components, while the gentleMACS™ Dissociator with Heaters is used for the mechanical dissociation steps during the on-instrument enzyme incubation. After the dissociation, the myelin and cell debris is removed using the Debris Removal Solution and is followed by a subsequent removal of erythrocytes using the Red Blood Cell Removal Solution (10×). The Anti-ACSA-2 MicroBead Kit is used to isolate astrocytes from the single-cell suspension.

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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).
  • D-PBS/BSA buffer: Prepare a solution containing D-PBS and 0.5% bovine serum albumin (BSA) by diluting MACS® BSA Stock Solution (# 130‑091‑376) 1:20 with D-PBS. Keep buffer cold (2−8 °C).
    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

  • AstroMACS Separation Buffer (# 130-117-336). Keep buffer cold (2−8 °C).
  • (Optional) Pre-Separation Filters (70 μm) (# 130-095-823
  • Anti-ACSA-2 MicroBead Kit, mouse (# 130-097-678)
  • Anti-ACSA-2 antibodies, mouse (clone IH3-18A3) conjugated to PE or APC. Learn more about our antibodies and dyes.
  • MACS Columns and MACS Separators: Anti-ACSA-2+ cell can be enriched by using MS Columns. Positive selection can also be performed by using the autoMACS Pro.
ColumnMax. number of labeled cellsMax. number of total cellsSelector
MS1x10⁷2x10⁷MiniMACS™, 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.
  • (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 (24 well) (# 130-098-263)
  • AstroMACS Medium (# 130-117-031) containing:
    MACS Neuro Medium
    MACS NeuroBrew®-21
    AstroMACS Supplement
  • 200 mM L-glutamine
  • Poly-L-Lysine (0.01%) and laminin
  • Penicillin/streptomycin

For immunocytochemical staining of cultured cells

  • Anti-GLAST (ACSA-1) pure, human, mouse, rat (# 130-095-822) and anti-rat-IgG2b secondary antibody or Anti-ACSA-2 pure, mouse (# 130-099-138) and anti-mouse-IgG2a secondary antibody
  • Staining buffer: Prepare a solution containing autoMACS Running Buffer (# 130-091-221) with FcR Blocking Reagent, mouse (# 130-092-575) in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.
  • Phosphate-buffered saline (PBS)
  • FcR Blocking Reagent, mouse (# 130-092-575)
  • autoMACS Running Buffer (# 130-091-221)
  • 2% paraformaldehyde (PFA) for fixation
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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.