20190503: ICC IgG and IgM HTB11 Neurons (CIDP+MS+small fiber PNP)

2019.04.25: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 –> 6 plates of 60 mm with 200.000 cells (with 26 coverslips)

2019.04.26: 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).

2019.05.03: ICC neuroblastoma neurons (day 8 of differentiation)

Samples:

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 for IgG and 1/40 for IgM (1h) –> 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

RESULTS: las neuronas no estaban demasiado bien (se despegan y se rompen). Repetir! (intentar mejorar el método)

Published by Cinta Lleixa