StemXVivo Neural Progenitor Differentiation Kit
R&D Systems, part of Bio-Techne | Catalog # SC035
Key Product Details
Assay Procedure
Refer to the product datasheet for complete product details.
Briefly, human pluripotent stem cells are differentiated into multipotent neural progenitor cells using the following differentiation procedure:
- Plate pluripotent stem cells on coated plates
- Replace Pluripotent Stem Cell Maintenance Media with NPC Differentiation Media
- Add fresh Complete NPC Differentiation Media daily for 7 days
- Evaluate differentiation using included SOX1 antibody
- Cells are ready for downstream applications
Reagents provided with the StemXVivo® Neural Progenitor Differentiation Kit (Catalog # SC035):
- NPC Differentiation Base Media Supplement (100X)
- NPC Differentiation Cocktail (1000X)
- Anti-Human SOX1 Antibody
Reagents
- DMEM/F12 (1X)
- BSA, very low endotoxin
- N-2 MAX Media Supplement (R&D Systems®, Catalog # AR009)
- GlutaMAX™ Supplement
- Penicillin-Streptomycin (optional)
- Phosphate Buffered Saline (PBS)
- Pluripotent Stem Cell Maintenance Media (MEF Conditioned Media; R&D Systems®, Catalog # AR005) or equivalent
- Cultrex® Stem Cell Qualified Reduced Growth Factor Basement Membrane Extract, Pathclear® (R&D Systems®, Catalog # 3434-001-02)
- Recombinant Human FGF basic (R&D Systems®, Catalog # 4114-TC or 233-FB)
- Y-27632 (Tocris®, Catalog # 1254)
- CryoDefend™-Stem Cells (R&D Systems®, Catalog # CCM018)
- Trypan Blue Solution
- Accutase® or Versene™
- 95% Ethanol
- 4% Paraformaldehyde in PBS
- 1% BSA in PBS
- 0.3% Triton™ X-100, 1% BSA, 10% normal donkey serum in PBS
- 1% BSA, 10% normal donkey serum in PBS
- Mounting Medium (R&D Systems®, Catalog # CTS011)
- Secondary developing reagent (R&D Systems®, Catalog # NL001)
- Deionized or distilled water
Materials
- Human pluripotent stem cells
- 24-well culture plates (or other, as needed)
- 60 mm culture plates
- 12 mm coverslips (optional)
- 15 mL and 50 mL centrifuge tubes
- 0.2 µm syringe filter
- 10 mL syringe
- Pipettes and pipette tips
- Serological pipettes
- Glass slides
- Fine pointed curved forceps
Equitpment
- 37 °C and 5% CO2 incubator
- 37 °C water bath
- Hemocytometer
- Centrifuge
- Inverted microscope
- Fluorescence microscope
Precaution: 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.
This protocol has been tested on cells cultured in pluripotent stem cell maintenance media (MEF Conditioned Media; R&D Systems®, Catalog # AR005) or equivalent. The quality and differentiation potential of human pluripotent stem cells at the onset of the differentiation protocol are of paramount importance to the efficiency of differentiation. Human pluripotent stem cells must be > 95% positive for Oct-3/4.
Coat wells with Stem Cell Qualified RGF BME.
Incubate at room temperature for 1-2 hours.
Day (-1) of Differentiation
Plate human pluripotent stem cells onto the coated plates at 1-2 x105 cells/cm2 in pluripotent stem cell maintenance media.
Culture cells to 80-90% confluency.
Day (0) of Differentiation
Replace the pluripotent stem cell maintenance media with Complete NPC Differentiation Media.
Incubate at 37 °C and 5% CO2 for 18-24 hours.
Days 1-7 of Differentiation
Replace the media with fresh Complete NPC Differentiation Media every 24 hours
Incubate at 37 °C and 5% CO2 for 18-24 hours.
Days 7 and Beyond
NPCs can be analyzed for neural progenitor cell markers, passaged onto new plates and cultured as NPCs, differentiated into downstream lineages, or cryopreserved.