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Mouse Oligodendrocyte Differentiation Kit

Key Product Details

Mouse Oligodendrocytes Generated Using the Mouse Oligodendrocyte Differentiation Kit. 
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SC004

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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