2019.03.19:seed cells (in proliferation medium)
- Coating with laminin 2’5 µg/ml diluted in Borate Buffer (for 1 h at 37ºC)
- Wash 2-3 times with PBS
- Seed aprox 10.000 cels/cm2 : 60 mm plate = 21 cm2
- In this case –> 1 plate of 60 mm with 250.000 cells (with 26 coverslips)
2019.03.20: differentiation
- Change all the medium of the cells (differentiation medium)
- Each time the new differentiation medium is changed, 10 μM retinoic acid (RA) is added –> stock solution -80ºC (diluted in ethanol 96 %): 0,01 M –> put 1 µl in every ml of differentiation medium (if we change the half of the medium, we have to add the double of RA)
- Once the differentiation has begun, change the half of the differentiation medium every 2 or 3 days (for 6 to 8 days).
2019.03.27: ICC neuroblastoma neurons (day 8 of differentiation)
| Sample | Result | Sample | Result |
| 19-010 | – | Zika W. 025 | – |
| 19-031 | – | Bochum 5 | – |
| 19-044 | – | Bochum 10 | – |
| 19-058 | – | Bochum 22 | – |
| ZN004 | – | Bochum 25 | – |
| ZN005 | – | Bochum 28 | – |
| ZN055 | – | Bochum 31 | – |
| ZN009 | – | Bochum 34 | – |
| ZN040 | – | Bochum 37 | – |
| Zika W. 003 | – | Bochum 38 | – |
| Zika W. 004 | – | Bochum 39 | – |
| Zika W. 005 | – | Neg. control | – |
| Zika W. 024 | – | 15-198 (CNTN1+) | + |
- Fix for 15 minutes with PFA 4 %
- Wash 3 times with PBS1x
- Block for 1 hour with Goat Serum 5 %
- Patient’s sera diluted 1/100 (1 h) –> in the box with parafilm, with the cells bellow
- Wash 3 times with PBS1x
- Secondary antibody: GAH488 diluted 1/1000 (1 h) –> in the box with parafilm, with the cells bellow
- Wash 3 times with PBS1x
- Vectashield mounting medium
RESULT: El control positivo se ve mal –> los axones están en mal estado (se han despegado durante la immuno, porque después de la fijación estaban todos pegados).
El resto de muestras se ven muy negativas (se ven núcleos, así que hay células).
Puede que el problema sea que los buffers se han hecho con agua destilada del grifo, y las sales que pueda llevar han hecho que se despeguen los axones. Hacer pruebas.
