Objective: to test the doble transfection of FLOT1 AND FLOT2 (with the same methodology of the paper that describes these autoantibodies)
2018.10.15
- Prepare 2 plates of 60mm of diameter with 14 coverslips of 12mm.
- Treatment with Poly-D-Lysina (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.
2018.10.16
[FLOT1] = 0,48 µg/µL. [FLOT2= 0,43 µg/µL.
D) 6 µg FLOT1 (25 µL) + 3 µg FLOT2 (14 µL) (+ 0’5 ml Optimem)
*For every plate of 60mm of diameter –> 12 microL of Lipofectamine. (+ 0’25 ml Optimem)
- 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.
2018.10.18 (48 hours later)
- Fix one plate with PFA 4%., and the other with acetone. 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: 1 coverslip of every plate.
- Blocking solution: 5% goat serum in PBS
- Ac anti-FLOT1: diluted1/50
- GAR 488 diluted 1/1000
- Ac anti-FLOT2 biotinilated: diluted 1/25
- Streptavidine 594 diluted 1/400
- Vectashield mounting medium
RESULT: both FLOT1 and FLOT2 are well transfected. There are no differences between the fixation with PFA 4 % and the fixation with Acetone.
