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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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.

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, human and mouse (# 130-093-634, # 130-093-636) or CD11b/c (Microglia) MicroBeads, rat (# 130-105-634, # 130-105-643)
  • 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
autoMACS®5×1071×108autoMACS Pro
Multi-242×1074×107MultiMACS Cell24
  • Fluorochrome-conjugated CD11b antibody, or CD11b/c antibody for flow cytometry analysis, e.g., CD11b-FITC, CD11b-PE, CD11b-APC, or CD11b/c-FITC, and CD45, e.g., CD45-FITC or CD45-APC. 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)
  • Mouse M-CSF (#130-094-129)

For immunocytochemical staining of cultured cells

  • CD11b pure, human and mouse and anti rat IgG2bκ secondary antibody or CD68 pure, mouse 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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