Differentiation of mature neurons from mouse NPCs

-Dilute Poly-L-Ornithine (PLO) 1:6 in H2O

-Fully coat the wells with diluted PLO (i.e. 500 ul per well in a 12WP)

-Leave in the incubator for minimum 2h (also overnight works)

-Aspirate the PLO and wash three times with sterile H2O

-Dilute Laminin 1:500 in sterile H2O

-Fully coat the wells with diluted Laminin (i.e. 500 ul per well in a 12WP)

-Leave in the incubator for minimum 2h (also overnight works)

-count the desired number of cells: Plate ~100.000 cells on a coverslip, ~150.000 cells in a 24WP well, ~250.000 cells in a 12WP well.

-seed NPCs for the final differentiation in NPC medium

-aspirate all NPC medium

-immediately add differentiation medium not directly on the bottom of the well but making it go through the walls to avoid disturbing the cells.

-Change medium (remove half volume and add half volume of new medium) avoiding the cells’ contact with air every 2-3 days. If at diff day 2-3 you notice overgrowth of glial cells, add AraC 1uM to the media in order to stop proliferation.

Cells can be kept in colture for >14 days, but they are already MAP2+ and TUJ1+ at day 7.

-Add 2.5 uM Tamoxifen to the medium, and the corresponding volume of metOH as control. Incubate for at least 6 days.