Neural differentiation of AAVS1-TRE3G-NGN2 pluripotent stem cells
Jiuchun Zhang, Harper JW
Abstract
This protocol is about neural differentiation of AAVS1-TRE3G-NGN2 pluripotent stem cells.
Before start
Prepare ND1 Medium and ND2 Medium:
ND1 Medium
DMEM/F12
N2 (100x) 1x
BDNF 10 ng/ml
NT3 10 ng/ml
NEAA (100X) 1x
Laminin 0.2 μg/ml
Doxycycline 2 μg/ml
ND2 Medium
Neurobasal medium
B27 (50x) 1x
Glutamax (100x) 1x
BDNF 10 ng/ml
NT3 10 ng/ml
Doxycycline 2 μg/ml
Attachments
Neural_differentiation_of_AAVS1-TRE3G-NGN2-pluripotent_stem_cells.pdf
Steps
Day 0:
Treat H9 AAVS1-TRE3G-NGN2 cells with Accutase and plate the dissociated cells in matrigel-coated 6-well plates (2x105 cells/well) in ND1 Medium supplemented with Y27632 (10micromolar (µM)).
Day 1:
Replace the medium with ND1 Medium .
Day 2:
Replace the medium with ND2 Medium .
Day 4:
Exchange 50% of the medium from each well.
Day 6:
Treat the cells with Accutase and replate the dissociated cells in matrigel-coated 6-well plates (2-4x105 cells/well) in ND2 Medium .
Day 8 and thereafter:
Exchange 50% of the medium from each well every other day.
