PROTOCOL: Neuroblastoma cells differentiation and ICC (SH-SY5Y)

Day 0: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 10.000 cels/cm²
- 100 mm plate = 58 cm²
- 60 mm plate = 21 cm²
- 35 mm plate = 9 cm²

Day 1: 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 5 to 7 days).

Proliferation medium (250 ml):	Differentiation medium (50 ml):
108’75 ml DMEM 108’75 ml F12 25 ml FBS (10%) 2’5 ml Pen-Strep (1 %) 2’5 ml L-glutamine (1 %) 2’5 ml Sodium pyruvate (1%)	48 ml Neurobasal 1 ml B27 (2 %) 25 µl NGF (50 ng/ml) 0’5 ml Pen-Strep (1 %) 0’5 ml Glutamax (1 %)

ICC neuroblastoma neurons

All the ICC is performed in a box with parafilm, with the cells above

Fix for 15 minutes with PFA 4 %
Wash 3 times with PBS1x
Block for 1 hour with Goat Serum 5 %
Patient’s sera
- For IgG: diluted 1/100 in Goat serum 5 % (1 h)
- For IgM: diluted 1/40 in Goat serum 5 % (1h)
Wash 3 times with PBS1x
Commercial antibody: anti-NF (AF3235) diluted 1/200 in Goat serum 5% (1h)
Wash 3 times with PBS1x
Secondary antibodies: GAC594 IgG + GAH488 (IgG or IgM) diluted 1/500 in Goat serum 5 % (1 h)
Wash 3 – 6 times with PBS1x (NO H2Od)
Vectashield mounting medium

Pruebas que se han hecho previamente y no han funcionado (las céls se despegan, o la tinción es muy ténue): (Cinta)

Cels HTB11:
- Anteriormente: cubres hacia arriba, y cubres hacia abajo
- 23.05.19: cubres en placa de 24 wells
- 24.05.19: montar con DAPI vs montar con Fluoromount (con y sin Hoescht)
- 31.05.19: 8-well culture slide (354632-corning) con Geltrex Matrix (coating). Se decide usar SH-SY5Y (crecen y diferencian mucho mejor y más rápido).
Cels SH-SY5Y:
- 31.05.19: 8-well culture slide (354632-corning) con Geltrex Matrix (coating)
- 31.05.19: immunos normales con cubres hacia arriba
- 17.06.19: placa 24 well –> céls crecidas directamente en pocillos, immuno in vivo vs fijadas, con hoescht
- 19.06.19: placa 24 well –> céls crecidas directamente en pocillos, immuno fijadas, sin hoescht
- 01.07.19, 02.07.19, 03.07.19: immunos normales con cubres hacia arriba
- 03.07.19: se descubre que el problema son los últimos lavados con H2O destilada –> no lavar al final con agua, sólo con PBS1x.
- A partir de este momento, el protocolo ha quedado establecido, y se han empezado a pasar todas las muestras.

Published by Cinta Lleixa