Mouse Oligodendrocyte Differentiation Kit
R&D Systems, part of Bio-Techne | Catalog # SC004
Key Product Details
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Summary for Mouse Oligodendrocyte Differentiation Kit
A kit optimized to efficiently differentiate mouse pluripotent cells into oligodendrocytes under serum-free conditions.
Key Benefits
- Pre-optimized to reduce experimental variation
- Defined conditions decrease variation
- High lot-to-lot consistency decreases variation
Why Use Pre-optimized Reagents for Differentiation of Pluripotent Stem Cells into Oligodendrocytes?
Successful differentiation of pluripotent stem cells into oligodendrocytes is dependent on high quality media and differentiation supplements.
Oligodendrocytes are cells of the central nervous system which myelinate axons and support rapid nerve conduction. In CNS disorders, such as stroke, multiple sclerosis and spinal cord injury, demyelination of axons contributes to functional deficits. Studies have demonstrated that neural transplantation therapy can restore functions lost by demyelination. However, the use of neural transplantation is limited by an inadequate supply of oligodendrocyte precursors. R&D Systems offers the Mouse Oligodendrocyte Differentiation Kit to drive successful and efficient differentiation of embryonic and induced pluripotent stem cells into oligodendrocytes.
R&D Systems Mouse Oligodendrocyte Differentiation Kit:
- Is pre-optimized to reduce variation.
- Has defined components to decrease variation.
- Includes sufficient reagents for the differentiation of 3 x 107 embryonic stem cells.
The Mouse Oligodendrocyte Differentiation Kit contains ITS and N-2 MAX Media Supplements, which are used to select and enrich neural precursor populations that are characterized by Nestin and A2B5 staining. Purified Bovine Fibronectin is included to support cell attachment and spreading. A growth factor panel, consisting of Recombinant Human Fibroblast Growth Factor Basic (FGF basic), Recombinant Human Epidermal Growth Factor (EGF), and Recombinant Human Platelet-derived Growth Factor AA (PDGF-AA) is included for effective oligodendrocyte differentiation.
The quantity of each component provided in the kit is estimated to be sufficient for the differentiation of 3 x 107 embryonic stem cells.
- Recombinant Human EGF
- Recombinant Human FGF basic
- Recombinant Human PDGF-AA
- Bovine Fibronectin
- ITS Media Supplement
- N-2 MAX Media Supplement
Stability and Storage
- Unopened Kit should be stored at = -20 °C in a manual defrost freezer. Do not use the kit past the expiration date.
- Opened Reagents can be stored at 2 °C to 8 °C for up to 1 month or aliquotted and stored at = -20 °C in a manual defrost freezer for up to 6 months.* Avoid repeated freeze-thaw cycles.
*Provided this is within the expiration date of the kit.
Limitations of the Procedure
- FOR LABORATORY RESEARCH USE ONLY. NOT FOR DIAGNOSTIC USE.
- The safety and efficacy of this product in diagnostic or other clinical uses has not been established.
- The quality of the embryonic stem cells and any variation in this procedure may cause variation in the efficiency of oligodendrocyte generation.
Precautions
- The acute and chronic effects of overexposure to reagents of this kit are unknown. Safe laboratory procedures should be followed and protective clothing should be worn when handling kit reagents.
- The ITS and N-2 MAX Supplements contain human transferrin. This transferrin was tested at the donor level using an FDA licensed method and found to be non-reactive for anti-HIV-1/2 and Hepatitis B surface antigen. As no testing can offer complete assurance of freedom from infectious agents, these reagents should be handled as if capable of transmitting infection.
Neural stem cells provide an excellent model for research focused on neural development and neurological disorders. R&D Systems offers ready-to-use primary cortical stem cells isolated from E14.5 Sprague-Dawley rats. In addition, primary mouse cortical stem cells isolated from E14.5 CD-1 mice are available. Every lot of R&D Systems Cortical Stem Cells is validated for a high level of Nestin expression and the capacity for multi-lineage differentiation into astrocytes, neurons, and oligodendrocytes. Our cortical stem cells are tested to ensure highest quality and lot to lot consistency. Both rat and mouse Cortical Stem Cells can be optimally expanded as monolayers or neurospheres.
To complement the use of primary neural stem cells, we offer a range of supportive products, including culture media which is specifically optimized for use with neural stem cells. We offer kits to promote the in vitro proliferation of neural precursors, and kits to differentiate neural stem cells into dopaminergic neurons or oligodendrocytes. In addition, kits are available which contain panels of antibodies designed to monitor the differentiation and identification of neural precursors, astrocytes, neurons, and oligodendrocytes.
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Mouse Oligodendrocytes Generated Using the Mouse Oligodendrocyte Differentiation Kit. D3 mouse embryonic stem cells were expanded in KO-ES Media and differentiated into oligodendrocytes using the Mouse Oligodendrocyte Differentiation Kit (Catalog # SC004). Oligodendrocytes were detected using a Mouse Anti-Human/Mouse/Rat/Chicken Oligodendrocyte Marker O4 Monoclonal Antibody (Catalog # MAB1326). The cells were stained with the NorthernLights™-557 Affinity-purified Goat Anti-Mouse Secondary Antibody (Catalog # NL019). The nuclei were counterstained with DAPI. View our protocol for Fluorescent ICC Staining of Stem Cells on Coverslips. |
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 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
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
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..
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