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Astrocyte Differentiation Protocol

This protocol utilizes the Astrocyte Differentiation Medium (Millipore cat. no. SCM010), a specially formulated medium optimized for the preferential differentiation of rodent neural stem cells to an astrocyte lineage. The medium has been extensively validated on mouse cortical and spinal cord neural stem cells and on rat hippocampal neural stem cells. Sufficient medium is supplied to provide for 100 separate reactions at 1 mL volume or 10 reactions at 10 mL volume.

Immunofluorescent images of neural stem cells differentiated with astrocyte differentiation medium (Millipore cat. no. SCM010): Adult Rat Hippocampal Neural Stem Cells (Millipore cat. no. SCR022) differentiated for 6–9 days in Astrocyte Differentiation Medium (Millipore cat. no. SCM010). Over 90% GFAP-positive cells (Millipore cat. no. AB5804) were detected with <1% MAP2-positive neurons and <5% O1-positive oligodendrocytes. (MAP2 and O1 staining not shown.)


Kit Components

  • 100 mL of Astrocyte
    Differentiation Medium
  • 100 µL of FGF-2,
    100 µg/mL stock


Additional Materials Required

  • Rodent neural stem cells — Millipore cat. nos. SCR022, SCR029, SCR031
  • Neural Stem Cell Characterization Kit — Millipore cat. no. SCR019
  • Neural Stem Cell Basal Medium — Millipore cat. no. SCM003
  • Rat/Mouse Neural Stem Cell Expansion Medium — Millipore cat. no. SCM008, SCM009
  • Poly-L-ornithine — Sigma cat. no. P3655
  • Laminin — Sigma cat. no. L-2020
  • Accutase — Millipore cat. no. SCR005
  • Hemocytometer
  • EGF (>107 units/mg) — Millipore cat. no. GF001
  • Heparin — Sigma cat. no. H-1027


Protocol

  1. Plate approximately 50,000 neural stem cells per well in a poly-L-ornithine/laminin coated 8-well chamber slide in Neural Stem Cell Basal Medium (Millipore cat. no. SCM003) containing the appropriate concentration of growth factors*. Use 0.75 mL total volume per well in the 8-well chamber slide to ensure that the cells are evenly distributed over the surface of the well.
  2. Incubate the cells at 37°C in a 5% CO2 humidified incubator. Allow the neural stem cells to settle and adhere to the slide before proceeding to step three. Usually the minimum amount of time is three or four hours post-plating. Do not exceed one day post-plating before proceeding to step three.
  3. Carefully aspirate the medium from each well. Cells are lightly adherent and caution must be applied to avoid aspirating the cells during this step.
  4. Apply 0.5 mL to 0.75 mL of the Astrocyte Differentiation Medium (pre-warmed to 37°C) to each well. Incubate at 37°C in a 5% CO2 humidified incubator.
  5. Replace with fresh Astrocyte Differentiation Medium every other day for six days.
  6. After the sixth day, replace with fresh Astrocyte Differentiation Medium containing 20 ng/mL FGF-2. This step helps induce the growth of astrocyte processes. For example: To make 6 mL astrocyte differentiation medium for one 8-well chamber slide, add 1.2 µL of the provided FGF-2 stock to 6 mL of the Astrocyte Differentiation Medium. Apply 0.75 mL of the mixture to each well of an 8-well chamber slide.
  7. Incubate at 37oC for an additional 2–3 days before fixing the culture with 4% paraform-aldehyde for antibody staining analysis. ReNcell Immortalized Progenitors and Serum-Free Medias

*For rat NSC, use 20 ng/mL FGF-2. For mouse neural stem cells, use 20 ng/mL FGF-2, 20 ng/mL EGF, and 2 µg/mL heparin.

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