2019.03.28: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 –> 2 plates of 60 mm with 200.000 cells (with 26 coverslips)
2019.03.29: 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).
2019.04.04: ICC neuroblastoma neurons (day 7 of differentiation)
Samples:
| Sample | Result | Sample | Result |
| 19-010 (IgG) | – | 18-512 (IgM) | – |
| 19-031 (IgG) | – | 18-513 (IgM) | – |
| 19-044 (IgG) | – | 18-514 (IgM) | – |
| 19-058 (IgG) | – | 18-515 (IgM) | – |
| ZN004 (IgG) | – | 18-516 (IgM) | – |
| ZN005 (IgG) | – | 18-517 (IgM) | – |
| ZN0055 (IgG) | – | 18-518 (IgM) | +++ |
| ZN009 (IgG) | – | 18-519 (IgM) | – |
| ZN040 (IgG) | – | 18-520 (IgM) | – |
| Zika 003 (IgG) | – | 18-521 (IgM) | – |
| Zika 004 (IgG) | – | 18-522 (IgM) | – |
| Zika 005 (IgG) | – | 18-523 (IgM) | – |
| Zika 024 (IgG) | – | 18-524 (IgM) | – |
| Zika 025 (IgG) | – | Neg. control (IgM) | – |
| 178-21 (IgG) | – | Neg. control (IgG) | – |
| 18-575 (IgG) | – | 142-2 (JGV) IgG | – |
| 18-509 (IgM) | – | 154-18 (AVP) IgG | +++ |
| 18-510 (IgM) | – | SH-SY5Y diferenciades: 142-2 (JGV) IgG | – |
| 18-511 (IgM) | – | SH-SY5Y diferenciades: 15-198 (AVP) IgG | +++ |
- 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 above
- Wash 3 times with PBS1x
- Secondary antibody: GAH488 IgG or IgM diluted 1/1000 (1 h) –> in the box with parafilm, with the cells above
- Wash 3 times with PBS1x
- Vectashield mounting medium
RESULT: la muestra de J.Gracia Vicens 18-518 se ve muy positiva también por IgM. Se ha testado una muestra de JGV de otro día y no es positiva por IgG.
El control positivo con las células SH-SY5Y diferenciadas se ve muy bien.
