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Human/Mouse Dopaminergic Neuron Differentiation Kit

Key Product Details

Characterization of Dopaminergic Neurons Generated from Human Pluripotent Stem Cells.
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SC001B

Assay Procedure

Refer to the product datasheet for complete product details.

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

  • Generate embryoid bodies from pluripotent cells
  • Select for and expand Nestin positive cells
  • Differentiate Nestin positive cells into dopaminergic neurons
 

 

Reagents Provided

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

  • ITS Supplement - 5 mL of a 100X concentrated solution, containing Bovine Insulin, Human Transferrin, and Sodium Selenite.
  • N-2 MAX Supplement - 5 mL of a 100X concentrated solution, containing Bovine Insulin, Human Transferrin, Sodium Selenite, Putrescine, and Progesterone.
  • Bovine Fibronectin Stock - 1 vial containing 2 mL of a 1000X (1 mg/mL) solution of purified Bovine Fibronectin.
  • Human FGF basic - 1 vial of lyophilized Recombinant Human FGF basic; enough to make 250 μL of a 1000X stock.
  • Mouse FGF-8b - 1 vial of lyophilized Recombinant Mouse FGF-8b; enough to make 250 μL of a 1000X stock.
  • Mouse Shh-N - 1 vial of lyophilized Recombinant Mouse Shh-N; enough to make 250 μL of a 1000X stock.

 

Other Supplies Required

Reagents

  • 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
  • Ascorbic Acid (Catalog # 4055/50)
  • Sterile, deionized water
  • BSA, very low endotoxin
  • Acetic acid
  • Anti-Nestin antibody (Catalog # AF2736 or IC1259)
  • Anti-Tyrosine Hydroxylase antibody (Catalog # MAB7566 or Catalog # AF7566)
  • Anti-Neuron-specific beta-III Tubulin antibody (Catalog # NL1195V or Catalog # MAB1195)

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 syringe filter
  • 0.2 μm, 500 mL filter units
  • Cryotubes
  • Serological pipettes
  • Pipettes and pipette tips
  • 10 mL syringes

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

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

Replace the KO-ES Media with ITS/Fibronectin Media

Replace the KO-ES Media with ITS/Fibronectin Media.

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

Replace the media every 2 days.

Verify successful differentiation by staining cells for Nestin.

Replace the KO-ES Media with ITS/Fibronectin Media

Expansion of Nestin-Positive Cells

 

Wash the cells twice with sterile PBS.

Dissociate the cells with 0.05% Trypsin/EDTA.

Add 5 mL of KO-ES Media to neutralize the Trypsin

Wash the cells twice with sterile PBS

Transfer the cells to a 15 mL tube.

Remove the cell clumps by allowing the tube to stand for approximately 5 minutes and then transferring the suspended cells to a 15 mL tube.

Transfer the cells to a 15 mL tube

Centrifuge the samples at 220 x g for 5 minutes.

Resuspend the cell pellet in N-2 MAX/FGF basic/FGF-8b/Shh-N/Ascorbic Acid Media.

Centrifuge the samples at 220 x g for 5 minutes

Perform a cell count.

Perform a cell count

Plate the cells on Poly-L-ornithine/Fibronectin-coated plates at 3-5 x 105 cells/well in 500 μl of media.

Replace the media daily for 4-6 days with N-2 MAX/FGF basic/FGF-8b/Shh-N/Ascorbic Acid Media.

Plate the cells on Poly-L-ornithine/Fibronectin-coated plates at 3-5 x 105 cells/well in 500 µl of media

Differentiation of Nestin-positive Cells to Dopaminergic Neurons

 

Culture the cells in N-2 MAX/Ascorbic Acid Media without growth factors for 10-15 days.

Replace the media every 2 days.

Culture the cells in N-2 MAX/Ascorbic Acid Media without growth factors for 10-15 days

After 10-15 days, dopaminergic neurons can be identified by staining for expression of Tyrosine Hydroxylase and Neuron-specific beta-III Tubulin.

After 10-15 days, dopaminergic neurons can be identified by staining for expression of Tyrosine Hydroxylase and Neuron-specific beta-III Tubulin

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FAQs

  • Can the Human/Mouse Dopaminergic Neuron Differentiation Kit work with a starting population of neural progenitor cells instead of mouse pluripotent stem cells?

    The kit may work with a starting population of neural progenitor cells, but we have not tested this. If starting with neural progenitor cells we recommend starting at Stage 5 of the Dopaminergic Neuron Differentiation Kit protocol. The starting cell density would also have to be optimized.

Product Documents

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