Objective: to test the doble transfection of flotillin 1 and flotillin 2.
Day 1- 2018.08.27:
- Prepare 1 plates of 60mm of diameter with 14 coverslips of 12mm.
- Treatment with Poly-D-Lysina (diluted 1/40 with PBS)
- 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 1.100.000 of cells.
- Incubate at 37ºC for 24h.
Day 2- 2018.08.28:
[FLOT1] = 0,48 µg/µL. [FLOT2]= 0,43 µg/µL.
6µg of FLOT 1 (15,5 µL) + 3µg of FLOT 2 (6,93 µL)
12 microL of Lipofectamine.
- Incubate at RT for 5 min.
- Unify every DNA with the appropriate amount of Lipo and incubate at RT for 30 min.
- For every plate: 5ml of HEK medium and 500microL of every mix of DNA and Lipo.
- Incubate O/N at 37ºC.
Day 3- 2018.08.29:
- 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: 2 coverslips
- Blocking solution: 5% goat serum in PBS
- Primary antibody:
- 1 coverslip: anti-FLOT1 diluted 1/100
- 1 coverslip: anti-FLOT2 diluted 1/200
- Secondary antibody: GAR488 diluted 1/1000
- Vectashield mounting medium
RESULT: FLOT1 and FLOT2 are well transfected, with this new condition we see higher amount of FLOT1 than previously, but still FLOT2 is predominant.
