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).
2019.04.03: ICC neuroblastoma neurons (day 6 of differentiation)
Objective: we want to test which are the best conditions to do the ICC. We try with 2 secondary antibodies (GAH488 or GAH594), with the buffers done with H2Od from bottle, and doing the incubations in 24 well plates/box with parafilm and the cells above/box with parafilm and the cells bellow.
Conditions:
| 24well-plate | box with parafilm (cells above) | box with parafilm (cells below) |
| 15-198 (GAH488) | 15-198 (GAH488) | 15-198 (GAH488) |
| Neg.ctrol (GAH488) | Neg.ctrol (GAH488) | Neg.ctrol (GAH488) |
| 15-198 (GAH594) | 15-198 (GAH594) | 15-198 (GAH594) |
| Neg.control (GAH594) | Neg.ctrol (GAH594) | Neg.ctrol (GAH594) |
| Ac anti-CNTN1 (RAG488) | Ac anti-CNTN1 (RAG488) | Ac anti-CNTN1 (RAG488) |
- 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)
- Wash 3 times with PBS1x
- Secondary antibody: GAH488/GAH594/RAG488 diluted 1/1000 (1 h)
- Wash 3 times with PBS1x
- Vectashield mounting medium
RESULT: the bests conditions are:
- H2Od from bottle
- ICC with the cells above (in box with parafilm)
- GAH488
- 6 – 8 days of differentiation
