2019.01.02 Morning:
- Prepare 3 plates of 60mm of diameter with 24 coverslips of 9mm.
- Treatment with Poly-D-Lysine (diluted 1/40 with boratte buffer)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS
- Trypsin-EDTA a plate of HEK293.
- For every plate of 60mm: 5ml of HEK medium + the volume that correspond to 1.000.000 of cells.
- Incubate at 37ºC for 24h.
2019.01.03 Afternoon:
Prepare the DNA and lipofectamine:
- [PLEXIN D1] = 0,46 µg/µL.
- 8 µg PLEXIN-D1 (17,4µl) + 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.
2019.01.04 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.
- Remove the blocking solution and freeze at -80ºC.
Check the transfection – ICC:
- Blocking solution: rabbit serum 1:40 in PBS
- Ac anti-PLEXIN-D1: diluted 1/1000
- RAG 488 IgG diluted 1/1000
- Vectashield mounting medium
Result: cells were well transfected with Plexin-D1
