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.

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
Matching products:

Protocol


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This protocol includes detailed instructions for preparing necessary reagents and materials and then carrying out each step.

Preparation of buffer for cell isolation and flow cytometry

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 1:20 with PBS. Keep buffer cold (2−8 °C). Prepare fresh on the day of use and degas the buffer, 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).

Preparation of cell culture plates

Coat a 24-well culture dish with 0.01% Poly-L-lysine overnight at 37 °C. Wash three times with ddH₂O the following day.

Preparation of medium for cell culture

Prepare the following cell culture medium: DMEM containing 10% FBS, 1% penicillin/streptomycin, and 2mM L-glutamine.

Use the Neural Tissue Dissociation Kit (T) or the Neural Tissue Dissociation Kit (P) to dissociate brain tissue and prepare a single-cell suspension. Follow the protocol of the kit data sheet.

Download data sheet

Neural Tissue Dissociation Kits

Good to know

The data sheet for the Neural Tissue Dissociation Kits includes a set of tips & hints to improve the quality and yield of the dissociation procedure. Refer to the data sheet appendix if, for example, the yield of viable cells is too low or a cell pellet will not form.

Isolate microglia from the single-cell suspension using either the CD11b (Microglia) MicroBeads, human and mouse or the CD11b/c (Microglia) MicroBeads, rat. Follow the protocol of the corresponding kit data sheet.

Download data sheet

CD11b (Microglia) MicroBeads, human and mouse

CD11b/c (Microglia) MicroBeads, rat

CD11b (Microglia) MicroBeads, human and mouse

CD11b+ microglia isolated from neonatal mouse brain tissue. CD11b+ cells were isolated from mouse neural cell suspension using the CD11b (Microglia) MicroBeads, an MS Column, and a MiniMACS™ Separator. Cells were fluorescently stained with CD11b-APC and CD45-FITC. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

CD11b/c (Microglia) MicroBeads, rat
Before MicroBead labeling
Before isolation
Negative fraction
Isolated CD11b/c+ cells

CD11b/c+ microglia isolated from neonatal rat brain tissue. CD11b/c+ cells were isolated from P3 rat brain tissue using the Neural Tissue Dissociation Kit (P), the gentleMACS™ Dissociator, CD11b/c (Microglia) MicroBeads, a MiniMACS™ Separator, and two MS Columns. Cells were fluorecently stained with CD11b/c-FITC in combination with CD45-APC and analyzed by flow cytometry using the MACSQuant® Analyzer. Cell debris and dead cells were excluded from the analysis based on scatter signals and propidium iodide fluorescence.

Analysis of mouse microglia

Notes:

  • The recommended antibody dilution for labeling cells is 1:10 for up to 1×107 cells/100 μL of PB buffer.
  • Volumes given below are for up to 1×107 nucleated cells. When working with fewer than 1×107 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g., for 2×107 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
  1. Use 100 μL of the CD11b+ fraction for analysis. Optionally, also analyze 100 μl of the negative fraction and 20 μL of the original fraction.
  2. Resuspend up to 1×107 nucleated cells per 100 μL of PB buffer (see "Things to prepare in advance of cell isolation and cell culture").
    ▲ Note: Always use freshly prepared buffer.
  3. Add 10 μL of CD11b-PE or other selected conjugated antibody.
  4. Mix well and incubate for 10 minutes in the refrigerator (2−8 °C) in the dark.
    Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
  5. Wash cells by adding 1 mL of PB buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
  6. Resuspend cell pellet in a suitable amount of PB buffer for analysis by flow cytometry, e.g., using the MACSQuant® Analyzer 10.

Analysis of rat microglia

Notes:

  • The recommended antibody dilution for labeling cells is 1:10 for up to 1×106 cells/50 μL of buffer.
  • Volumes given below are for up to 1×106 nucleated cells. When working with fewer than 1×106 cells, use the same volumes as indicated. When working with higher cell numbers, scale up all reagent volumes and total volumes accordingly (e.g., for 2×106 nucleated cells, use twice the volume of all indicated reagent volumes and total volumes).
  1. Use 100 μL of the CD11b/c+ fraction for analysis. Optionally, also analyze 100 μl of the negative fraction and 20 μL of the original fraction.
  2. Resuspend up to 1×106 nucleated cells per 45 μL of buffer.
    ▲ Note: Always use freshly prepared buffer.
  3. Add 5 μL of CD11b/c-PE or other selected conjugated antibody.
  4. Mix well and incubate for 10 minutes in the refrigerator (2−8 °C) in the dark.
    Note: Higher temperatures and/or longer incubation times may lead to non-specific cell labeling. Working on ice requires increased incubation times.
  5. Wash cells by adding 1 mL of buffer and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
  6. Resuspend cell pellet in a suitable amount of buffer for analysis by flow cytometry, e.g., using the MACSQuant Analyzer 10.
  1. Plate 1×105 cells in 50 μL of pre-warmed prepared medium as a drop in the middle of each well of a coated 24-well plate (see "Things to prepare in advance of cell isolation and cell culture").
  2. Let cells settle down for 30 minutes at 37 °C in the incubator.
  3. Carefully add 450 μL of prepared medium to each well.
  4. Maintain the culture by replacing 50% of medium every other day.

Things to prepare in advance

Staining buffer: Prepare a solution containing autoMACS® Running Buffer with FcR Blocking Reagent, mouse  in a ratio of 1:10, e.g., add 1 mL FcR Blocking Reagent to 9 mL autoMACS Running Buffer.
  1. Wash cells 3× with PBS.
  2. Fix cells with 2% PFA for 10 minutes at room temperature.
  3. Wash cells 3× with PBS.
    Note: When working with CD68 antibodies, add 0.2% TRITON™ X-100 in PBS, incubate for 10 minutes at room temperature, and wash cells 3× with autoMACS Running Buffer.
    Note: Fixed cells can be stored in azide-containing buffer at 2–8 °C for up to 1 week.
  4. Add staining buffer (see "Things to prepare in advance") and incubate for 10 minutes at room temperature.
  5. Discard staining buffer.
  6. Add primary antibody in staining buffer to the cells with a final concentration of 5–10 μg/mL and incubate at room temperature in the dark for 10 minutes.
    When working with CD68 antibodies: Incubate at 2–8 °C overnight in the dark
  7. Wash cells 3× with autoMACS Running Buffer.
  8. Add a corresponding secondary antibody in staining buffer to the cells.
    When working with CD11b antibodies: Incubate at room temperature in the dark for 10 minutes.
    When working with CD68 antibodies: Incubate at room temperature in the dark for 1 hour.
  9. Wash cells 3× with autoMACS Running Buffer.
    Note: For co-staining with additional antibodies repeat steps 6–9.
  10. Store cells in autoMACS Running Buffer.
  11. Cells are now ready for immunofluorescence microscopy.
    Note: Samples can be stored at 2–8 °C in the dark for up to one week
    Note: When working with cells cultured on coverslips, the coverslips need to be mounted onto slides before imaging.
View details

Successful culture of microglia isolated from neonatal mouse brain tissue. Neonatal mouse whole brains were dissociated with the Neural Tissue Dissociation Kit (P). Subsequently, microglia were isolated using the CD11b (Microglia) MicroBeads. Microglia were cultured in DMEM, 10% FCS, 1% P/S and 2 mM L-glutamine on PLL-coated glass coverslips (1×105 cells per well of a 24-well plate). Cells were fixed and stained with the microglia-specific antibodies CD45 (A) and  CD11b (B).

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