Application protocol

Isolation and cultivation of microglia from neonatal mouse or rat brain

This application protocol describes the isolation of highly purified and viable microglia from neonatal brain tissue. Brain tissue from mice or rats younger than P8 is dissociated into single-cell suspensions via enzymatic digestion of the extracellular matrix and gentle mechanical dissociation. CD11b (Microglia) MicroBeads, mouse or CD11b/c (Microglia) MicroBeads, rat are used to isolate microglia from the single-cell suspension. CD11b (Microglia) MicroBeads isolate CD11b+ cells, whereas the CD11b/c (Microglia) antibody appears to recognize a common epitope shared between CD11b and CD11c. CD11b, also known as integrin alpha M (ITGAM) or Mac-1, is a component of complement factor 3 (CR3). CD11c, also called integrin alpha X (ITGAX), is a component of complement receptor 4 (CR4). CD11b and CD11c are expressed on microglia, macrophages, monocytes, granulocytes, NK cells, and dendritic cells.

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 (T) (# 130-093-231) or 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 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

  • CD11b (Microglia) MicroBeads, human and mouse (# 130-093-634, # 130-093-636) or CD11b/c (Microglia) MicroBeads, rat (# 130-105-634, # 130-105-543)
  • 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: Microglia can be enriched using MS or LS Columns, or depleted using LD Columns. Positive selection can also be performed using the autoMACS® Pro Separator or the MultiMACS™ Cell24 Separator. 
ColumnMax. number of labeled cellsMax. number of total cellsSeparator
Positive selection
MS1×10⁷2×10⁷MiniMACS™, OctoMACS™
LS2×10⁷4×10⁷MidiMACS™, QuadroMACS™
Depletion
LD1.5×10⁷3×10⁷MidiMACS, QuadroMACS
Positive selection or depletion
autoMACS5×10⁷ 1×108autoMACS Pro
Multi-242×10⁷4×10⁷MultiMACS Cell24
  • Fluorochrome-conjugated CD11b antibody, or CD11b/c antibody for flow cytometry analysis, e.g., CD11b-FITC (# 130-081-201), CD11b-PE (# 130-091-240), or CD11b-APC (# 130-091-241), or CD11b/c-FITC (# 130-105-273) and CD45, e.g., CD45-FITC (# 130-110-658), or CD45-APC (130-110-660). 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)
  • DMEM with stable glutamine
  • L-glutamine
  • Poly-L-lysine (0.01%)
  • Penicillin/streptomycin
  • Fetal bovine serum (FBS)

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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