Jun 28, 2023
Differentiation of mature neurons from mouse NPCs
- Sonia Cruciani1,
- Eva Maria Novoa1
- 1Center for Genomic Regulation (CRG)
Protocol Citation: Sonia Cruciani, Eva Maria Novoa 2023. Differentiation of mature neurons from mouse NPCs. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4oo2gmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 21, 2022
Last Modified: June 28, 2023
Protocol Integer ID: 70334
Funders Acknowledgements:
MICINN Spanish Grant Funding
Grant ID: PID2021-128193NB-I00
Abstract
This is a step by step protocol used to differentiate mouse NPCs to mature neurons.
Materials
·Mouse NPCs: vial of mNPCs derived from mES mettl3#6Cre from TEF
·Neurobasal (Thermo scientific, cat. no. 21103-049)
·DMEM/F12 (Thermo scientific, cat. no. 11320-033 )
·N2 supplement (Gibco, cat. no. 17502-048)
·Phosphate-buffered saline (PBS; Life technologies, cat. no. 14190-169)
·EDTA 0.5M pH 8
·B27 minus vitamin A (Gibco, cat. no. 11500446)
·Non-essential amino acids (Thermo scientific, cat no.11140-035)
·2-mercaptpethanol (Thermo scientific, cat no.31350-010)
·Glutamax (life technologies, cat no. 35050-061)
·P/S (Thermo scientific, cat no. 15140-122)
·Laminin (Sigma cat no. L2020)
·Poli-L-Ornithine solution (Sigma cat no. A004-C)
·EGF (Gibco, cat no. PMG3043)
·FGF2 (aka bFGF) (Stem cell technologies, cat no. 78003)
·DAPT (Selleck,cat no. S2215)
·BDNF (Sigma,cat no. B3795)
·FBS
·DMSO
●N2B27 medium:
| Component | Volume for 50ml | |
| Neurobasal | 24 mL | |
| DMEM/F12 | 24 mL | |
| P/S | 0.5 mL | |
| MEM Non-Essential Amino Acids Solution | 0.5 mL | |
| GlutaMAX™- 100X | 0.5 mL | |
| B-27 without vitamin A | 0.5 mL | |
| N2 | 0.25 mL | |
| β-mercaptoethanol, 1000X. Sigma M-7522 | 50 uL |
This medium could be stored 1 month at 4 C
●NPC medium:
N2B27 + EGF 10 ng/ml + FGF2 10 ng/ml
●differentiation medium:
N2B27 + BDNF 40 ng/ml + DAPT 10 uM
Coating
Coating
-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)
Plating - Day -1
Plating - Day -1
-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
Day 0
Day 0
-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.
Day 2-14
Day 2-14
-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.
Tamoxifen treatment
Tamoxifen treatment
-Add 2.5 uM Tamoxifen to the medium, and the corresponding volume of metOH as control. Incubate for at least 6 days.