Application protocol

Isolation and cultivation of microglia from adult mouse or rat brain

In this application protocol, we generate highly purified and viable microglia from adult mouse or rat brain tissue. Brain tissue from mice or rats 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 are removed using the Debris Removal Solution and is followed by subsequent removal of erythrocytes using the Red Blood Cell Removal Solution. The CD11b (Microglia) MicroBeads, mouse and human or the CD11b/c (Microglia) MicroBeads, rat are used to isolate microglia 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).
  • 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.
    Note: 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 or rat serum albumin, mouse or rat 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

  • CD11b (Microglia) MicroBeads, mouse (# 130-093-634, # 130-093-636)
  • CD11b/c (Microglia) MicroBeads, rat (# 130-105-634, # 130-105-543)
  • (Optional) Pre-Separation Filters (70 µm) (# 130-095-823)
  • MACS Columns and MACS Separators: microglia can be enriched using MS or LS Columns. Positive selection can also be performed using the autoMACS Pro Separator. 
ColumnMax. number of labeled cellsMax. number of total cellsSelector
MS1 x10⁷2 x10⁷MiniMACS™, OctoMACS™,
VarioMACS, SuperMACS II
LD2 x10⁷ 4 x10⁷MidiMACS™, QuadroMACS™,
VarioMACS, SuperMACS II
autoMACS5 x10⁷1 x108autoMACS 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. 
  • CD11b antibodies, human and mouse (clone M1/70.15.11.5), or CD45 antibodies, mouse (clone 30F11), or CD11b/c antibodies rat (clone REA325), or CD45 antibodies, rat (clone REA504) conjugated to PE or APC. Learn more about our antibodies and dyes.
  • (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)
  • DMEM with stable glutamine
  • 200 mM L-glutamine
  • Poly-L-lysine (0.01%)
  • Penicillin/streptomycin
  • FBS (fetal bovine serum)

For immunocytochemical staining of cultured cells

  • CD11b pure, human and mouse (# 130-115-811) and anti‑rat IgG2bκ secondary antibody or CD68 pure, mouse (# 130‑115‑808) and anti-rat 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
  • (Optional) 0.2% TRITON™ X-100 in PBS
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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.