Brn-3b siRNA (m)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 1 × 10^5 cells/well.	Culture cells in 2 ml differentiation medium supplemented with BDNF. Incubate cells for 72 h.

Upstream tips
Seed 1 × 10^5 cells/well.
Protocol tips
Culture cells in 2 ml differentiation medium supplemented with BDNF. Incubate cells for 72 h.

Publication protocol

The purified neurospheres were collected and dissociated with Accutase: Sigma, St. Louis, MO, USA, and the stem cells were seeded into six-well culture plates at a density of 1 × 105 cells/well, and cultured in 2 ml differentiation medium supplemented with BDNF (1 ng/ml), RA: retinoic acid (1 μM) and 1% FBS. The cells were randomly divided into groups as follows: Group a1: Brn-3b siRNA group (sc-38767), Group a2: Control siRNA group (scrambled sequence, sc-37007), Group a3: Control group (without any handling); Group b1: Isl-1 siRNA group (sc-37122), Group b2: Control siRNA group (scrambled sequence, sc-44230), Group b3: Control group (without any handling); Group c1: Notch signal pathway inhibitor (GSI) group (sc-221656), Group c2: Control group (without any handling). The stem cells were transfected with the indicated siRNA or treated with GSI. After 72 h, the cells were transfected with lentivirus PGC-FU- Atoh7-GFP. At seven days after transfection, immunofluorescence staining was performed to detect the percentage of ganglion cells in total differentiated cells.

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