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.

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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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: MACS Neuro Medium containing 2% MACS NeuroBrew®-21, 1% penicillin/streptomycin and 0.5 mM L-glutamine.

Use the Neural Tissue Dissociation Kit (P) or Neural Tissue Dissociation Kit (T) 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 the ACSA-2-positive astrocytes from the single-cell suspension using the Anti-ACSA-2 MicroBead Kit, mouse. Follow the protocol of the kit data sheet.

Download data sheet

Anti-ACSA-2 MicroBead Kit, mouse

Notes:

  • The recommended antibody dilution for labeling cells is 1:10 for up to 1×10⁶ cells/50 μL of PB 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 ACSA-2+ 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 PB buffer (see "Things to prepare in advance of cell isolation and cell culture").
    ▲ Note: Always use freshly prepared buffer.
  3. Add 5 μL of Anti-ACSA-2-APC, mouse antibodies.
  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.
  1. Plate 5×104 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 the 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: 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 1–5 μg/mL and incubate at room temperature in the dark for 10 minutes.
  7. Wash cells 3× with autoMACS Running Buffer.
  8. Add a corresponding secondary antibody in staining buffer to the cells and incubate at room temperature in the dark for 10 minutes.
  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 ACSA-2-positive astrocytes isolated from neonatal mouse brain tissue. P3 whole mouse brains were dissociated using the Neural Tissue Dissociation Kit (P) and astrocytes were isolated from the single-cell suspension using the Anti-ACSA-2 MicroBead Kit. Astrocytes were cultured in MACS® Neuro Medium, MACS NeuroBrew®-21, 1% P/S, and 0.5 mM L-glutamine, on PLL-coated glass coverslips (5×104 cells per well). After 3 days in culture, isolated astrocytes were fixed and stained with the astrocyte-specific antibodies Anti-GLAST (green) and Anti-GFAP (red).

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