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Mouse Oligodendrocyte Differentiation Kit

Key Product Details

Mouse Oligodendrocytes Generated Using the Mouse Oligodendrocyte Differentiation Kit. 

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, pluripotent stem cells are differentiated into oligodendrocytes using the following procedure:

  • Generate embryoid bodies from pluripotent stem cells
  • Select for and expand Nestin-positive cells
  • Induce A2B5-positive cells using supplemented media
  • Differentiate A2B5-positive cells into oligodendrocytes
 

 

Reagents Provided

Reagents Supplied in the Mouse Oligodendrocyte Differentiation Kit (Catalog # SC004).

  • Human Recombinant EGF -sufficient to make 100 μl of a 1000X stock solution
  • Human Recombinant FGF basic -sufficient to make 500 μl of a 1000X stock solution
  • Human Recombinant PDGF-AA -sufficient to make 100 μl of a 1000X stock solution
  • Bovine Fibronectin Stock -1 vial containing 2 mL of a 1000X (1 mg/mL) solution of purified Bovine Fibronectin
  • ITS Media Supplement -5 mL of a 100X concentrated stock solution containing Bovine Insulin, Human Transferrin, and Sodium Selenite
  • N-2 MAX Media Supplement -5 mL of a 100X concentrated stock solution containing Recombinant Human Insulin, Human Transferrin, Sodium Selenite, Putrescine, and Progesterone

 

Other Supplies Required

Reagents

  • Dulbecco’s Modified Eagle Medium (DMEM)
  • DMEM/F-12, no HEPES
  • Fetal Bovine Serum, ES Cell Qualified
  • Phosphate Buffered Saline (PBS)
  • 0.05% Trypsin/EDTA
  • Gelatin
  • ESGRO® (recombinant mouse LIF) (Millipore, or equivalent)
  • Knock-out DMEM
  • MEM Non-essential AA Solution
  • Penicillin-Streptomycin-Glutamine, 100X
  • Penicillin-Streptomycin, 100X
  • 2-Mercaptoethanol, 1000X
  • Glucose
  • L-Glutamine
  • Sodium Bicarbonate, NaHCO3
  • Poly-L-ornithine
  • T3 (3,3’,5-Triiodo-L-thyronine sodium salt)
  • Sterile, deionized water
  • BSA, very low endotoxin
  • Acetic acid

Materials

  • Mouse Embryonic Stem (ES) cells
  • Irradiated mouse embryonic fibroblast (iMEF) feeder cells (Catalog # PSC001)
  • 10 cm tissue culture dishes
  • 10 cm bacterial culture dishes
  • 12 mm cover slips
  • 24-well culture plates
  • 15 mL centrifuge tubes
  • 0.2 μm, 500 mL filter units
  • 0.2 μm syringe filter
  • 10 mL syringes
  • Cryotubes
  • Serological pipettes
  • Pipettes and pipette tips

Equipment

  • 37 °C and 5% CO2 incubator
  • 37 °C water bath
  • 60 °C hot plate
  • Centrifuge
  • Hemocytometer
  • Microscope
 

 

Procedure Overview

Protocol for the Differentiation of Pluripotent Cells into Dopaminergic Neurons using the Human/Mouse Dopaminergic Neuron Differentiation Kit (Catalog # SC001B)

Selection of Nestin-positive Cells

Generate embryoid bodies (EB) from pluripotent stem cells.

Transfer the EB to a 10 cm culture dish containing KO-ES Media.

Culture the cells for 24 hours at 37 °C and 5% CO2.

Generate embryoid bodies (EB) from pluripotent stem cells

Replace the KO-ES Medium with 10 mL of ITS/Fibronectin Media.

Culture the cells for 6-8 days at 37 °C and 5% CO2.

Replace the ITS/Fibronectin Media every 2 days.

Verify successful differentiation by staining cells for Nestin.

Replace the KO-ES Medium with 10 mL of ITS/Fibronectin Media

Induction of A2B5-positive cells

 

Wash the attached cells twice with sterile PBS.

Dissociate the cells with 0.05% Trypsin/EDTA solution.

Add 5 mL of KO-ES Media.

Wash the attached cells twice with sterile PBS

Transfer the cells to a 15 mL tube

Remove the cell clumps (remnants of EBs) by allowing the tube to stand for about 5 minutes and then transferring the suspended cells to a 15 mL tube.

Transfer the cells to a 15 mL tube

Centrifuge the suspension for 5 minutes at 220 x g.

Resuspend the cell pellet in N-2 MAX/FGF Media.

Centrifuge the suspension for 5 minutes at 220 x g

Perform a cell count.

Perform a cell count

Plate the cells at 1 x 105 cells/well in 500 μL of N-2 MAX/FGF Media onto Poly-L-ornithine/Fibronectin coated plates.

Replace the media daily with:

  • N-2 MAX/FGF/EGF Media daily for 4 days.
  • N-2 MAX/FGF/EGF Media for the next 4 days.
  • N-2 MAX/FGF/PDGF-AA Media for the final 4 days.

Verify successful induction by staining cells for A2B5

Plate the cells at 1 x 105 cells/well in 500 µL of N-2 MAX/FGF Media onto Poly-L-ornithine/Fibronectin coated plates

Differentiation to Oligodendrocytes

 

Replace the media on A2B5 cells with N-2 MAX/T3 Media.

Replace the media every 2 days for 6-8 days.

Replace the media on A2B5 cells with N-2 MAX/T3 Media

Verify successful differentiation by staining cells for expression of Oligodendrocyte Marker O4..

Verify successful differentiation by staining cells for expression of Oligodendrocyte Marker O4

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