Application protocol

Isolation and cultivation of ACSA-2-positive astrocytes from neonatal mouse brain

 In this application protocol, we describe a protocol to generate highly purified and viable astrocytes from neonatal mouse brain tissue. Brain tissue from mice younger than P8 is dissociated into single-cell suspensions and astrocytes are then isolated using the Anti-ACSA-2 MicroBead Kit. The ACSA-2 antigen is expressed specifically on astrocytes in a pattern similar to GLAST, and serves as a specific marker for astrocytes in the central nervous system.1,2 The proportion of ACSA-2+ astrocytes in brain tissue differs according to mouse age and brain region used for cell isolation.

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) or Neural Tissue Dissociation Kit (T) (# 130-093-231)
  • 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 tube
  • 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)
  • C Tubes (# 130-093-237, # 130-096-334)
  • (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)

For cell isolation and flow cytometry analysis

  • Anti-ACSA-2 MicroBead Kit, mouse (# 130-097-678) or Anti-ACSA-2 MicroBead Kit, mouse - small (# 130-097-679)
  • Pre-Separation Filters (70 μm) (# 130-095-823)
  • 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.
    Note: BSA can be replaced by other proteins such as mouse serum albumin, mouse serum, or fetal bovine serum (FBS).
  • MACS Columns and MACS Separators: ACSA-2+ cells can be enriched by using MS or LS Columns or depleted with the use of LD Columns. Positive selection or depletion can also be performed by using the autoMACS® Pro Separator. 
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Positive selection
MS1×10⁷2×10⁷MiniMACS™, OctoMACS™,
VarioMACS, SuperMACS II
LS2×10⁷4×10⁷MidiMACS™, QuadroMACS,
VarioMACS, SuperMACS II
Depletion
LD1.5×10⁷3×10⁷MidiMACS, QuadroMACS,
VarioMACS, SuperMACS II
Positive selection or depletion
autoMACS5×10⁷ 1×108autoMACS 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 Anti-ACSA-2 antibodies for flow cytometry analysis, e.g., Anti-ACSA-2-APC (# 130-102-315). Learn more about our antibodies and dyes.
  • Propidium Iodide Solution (# 130-093-233) or 7-AAD for flow cytometry exclusion of dead cell
  • MACSQuant® Analyzer 10 (# 130-096-343)

For cell culture

  • Double-distilled water (ddH₂O)
  • Imaging Plate CG 1.5 (24 well) (# 130-098-263)
  • MACS Neuro Medium (# 130-093-570) 
  • MACS NeuroBrew®-21 (# 130-093-566)
  • L-glutamine
  • Poly-L-lysine (0.01%)
  • 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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