Mouse Oligodendrocyte Differentiation Kit
R&D Systems, Inc. a Bio-Techne Brand
Key Product Details
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 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
- 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
- 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
- Sodium Bicarbonate, NaHCO3
- T3 (3,3’,5-Triiodo-L-thyronine sodium salt)
- Sterile, deionized water
- BSA, very low endotoxin
- Acetic acid
- 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
- Serological pipettes
- Pipettes and pipette tips
- 37 °C and 5% CO2 incubator
- 37 °C water bath
- 60 °C hot plate
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.
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.
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.
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.
Centrifuge the suspension for 5 minutes at 220 x g.
Resuspend the cell pellet in N-2 MAX/FGF Media.
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
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.
Verify successful differentiation by staining cells for expression of Oligodendrocyte Marker O4..
Citations for Mouse Oligodendrocyte Differentiation Kit (1)
Citations are publications that use Bio-Techne products. Selected citations for Mouse Oligodendrocyte Differentiation Kit include:
Sample Types: Whole Cells
Applications: BioassayT Deng et al. (2016), Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation Nucleic Acids Res, 0(0).
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