Day 0- 2018.05.22: Coating and seeding
- Prepare 2 plates of 60mm of diameter with 14 coverslips (12 mm)
- Treatment with Poly-D-Lysine (diluted 1/40 with PBS)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS 1x
- Treatment with Laminin (diluted 2 microL/ml PBS)
- Wash with PBS 1x
- Seed 10000 cells/cm2 –> every plate has 21 cm2 –> 210000 cells/plate 60 mm (in proliferation medium)
- Incubate at 37ºC for 24h.
*Proliferation medium:
- DMEM/F12 (1:1)
- 10 % FBS
- 1 % Pen-Strep
- 1 % L-glutamine
- 1 % Sodium pyruvate
Day 1- 2018.05.23: treatment and medium exchange
Change the medium of the cells –> Add retinoic acid to the differentiation medium.
- Differentiation medium:
- Neurobasal –> 48 ml
- 2 % B27 (stock at 50x) –> 1 ml
- 1 % Glutamax –> 0’5 ml
- 50 ng/ml NGF (stock at 100 µg/ml) –> 25 µl
- 1 % Pen-Strep –> 0’5 ml
- Retinoic Acid (RA) 10 µM –> Stock solution -80ºC at 0,01 M –> 1 µl every ml of medium
Day 3- 2018.05.25: treatment and medium exchange
Same conditions as day 1
Day 6– 2018.05.28: treatment and medium exchange
Same conditions as day 1
Day 7- 2018.05.29: ICC (proof)
- Fix with PFA 4% (aprox 5 ml/plate). Incubation at RT during 10min.
- Wash with PBS 1x
- Blocking solution: Goat Serum 5 % in PBS for 1h at RT.
- Patient’s sera diluted 1/100 with 5 % Goat Serum.
- Wash with PBS 1x
- Secondary antibody: GAH488 IgG diluted 1/1000
- Wash with PBS 1x
- Vectashield mounting medium
Samples: the same samples as the day 22.05.18
Name | ID | IgG/IgM | Result | |
---|---|---|---|---|
Sorli Peña, Miguel A. | 18-517 | IgG | CIDP CNTN1 ++ for HTB11 (undifferentiated cells) | |
Gracia Vicens, JM | 18-518 | IgG | CIDP ++ for HTB11 (undifferentiated cells) | |
Jalabo Remacha, M.Carmen | 18-526 | IgG | CIDP + for HTB11 (undifferentiated cells) | |
Prado Pintor, Carmen | 18-561 | IgG | CIDP + for HTB11 (undifferentiated cells) | |
102 | IgG | EM + for HTB11 (undifferentiated cells) | ||
Cussó, Pere | 18-600 | IgG | +++ for DRG neurons (rat) | |
Cheng Swee Chin | 17-511 | IgG | ++ for DRG neurons (rat) | |
Simola Jari Olavi | 17-513 | IgG | + for DRG neurons (rat) | |
74.09649-SER | 18-596 | IgG | ++ for DRG neurons (rat) | |
V.Hilton Jane | 18-601 | IgG | - for DRG neurons (rat) | |
Conchillo Riera, Pere | 15-611 | IgG | NMM | |
Val Sánchez de León, Leonardo | 09-5162 | IgG | NMM | |
Marqués Mercadal, Joan Antoni | 196-13 | IgG | NMM | |
Negative control | IgG |
Results:
- Se ven muy pocas células, y los axones están todos despegados.
- Hay precipitado del Ac secundario