2019.07.29 Morning:
- Prepare 4 plates of 60mm of diameter with 24 coverslips of 9mm.
- Treatment with laminin 2,5 microl/ml diluted in boratte buffer
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS
- Trypsin-EDTA a plate of LEC1 cells
- For every plate of 60mm: 5ml of LEC1 medium + the volume that correspond to 500.000 of cells.
- Incubate at 37ºC for 24h.
2019.07.30 Afternoon:
Prepare the DNA and lipofectamine:
- 3 plates: 1) CNTN1, 2) Caspr1, 3) CNTN1/Caspr1 (Same protocol as transfection in HEK cells)
- 8 µg DNA+ 250 µl Optimem / plate
- 12 µL of Lipofectamine + 250 µl Optimem / plate
- Incubate at RT for 5 min.
- Unify DNA with Lipofectamine and incubate at RT for 30 min.
- Add 500microL of the mix of DNA and Lipofectamine to the plate.
- Incubate O/N at 37ºC.
2019.07.31 Morning:
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Blocking solution: Rabbit serum 1:40 in PBS for 1h at RT (CNTN1 and CNTN1/Caspr1), and Goat 5% for Caspr1 and CNTN1/Caspr1
- Remove the blocking solution and freeze at -80ºC.
Check the transfection – ICC:
CNTN1 and CNTN1/Caspr1:
- Blocking solution: rabbit serum 1:40 in PBS
- Ac anti-CNTN1 diluted 1/1000
- RAG 488 IgG diluted 1/1000
- Vectashield mounting medium
Result: cells were well transfected with CNTN1
Caspr 1, CNTN1/Caspr1:
- Blocking solution: goat 5% in PBS
- Ac anti-Caspr1 diluted 1/250
- GAR 488 IgG diluted 1/1000
- Vectashield mounting medium
Result:
All cells were well transfected. Cell did not detached during the ICC.
I think next time we can repeat this protocol with 650.000 cells.
