2018.12.11
- Prepare four 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 1.200.000 of cells.
- Incubate at 37ºC for 24h.
2018.12.12
Morning
Prepare the DNA and lipofectamine:
[FLOT1] = 0,48 µg/µL. [FLOT2]= 0,43 µg/µL –>
6 µg FLOT1 (12,5µl) + 3 µg FLOT2 (6,9) + 250 µl Optimem / plate
12 µL of Lipofectamine + 250 µl Optimem / plates
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.
2018.12.13
Afternoon
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Blocking solution: 5% goat serum in PBS for 1h at RT.
- Remove the blocking solution and freeze at -80ºC.
Check the transfection – ICC:
- Blocking solution: 5% goat serum in PBS
- Ac anti-FLOT1: diluted1/50
- GAR 488 IgM diluted 1/1000
- Ac anti-FLOT2 biotinilated: diluted 1/25
- Streptavidine 594 diluted 1/400
- Vectashield mounting medium
RESULT: both FLOT1 and FLOT2 are transfected, the cells look good, and there are very few dead cells.
