Day 1- 2018.02.12:
- Prepare 2 plates of 60mm of diameter with 12 coverslips of 12mm.
- Treatment with Poly-D-Lysina (diluted 1/40 with PBS 1x)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS 1x.
- Trypsin-EDTA a plate of HEK293.
- For every plate of 60mm: 5ml of HEK medium + the volume that correspond to 800.000 of cells.
- Incubate at 37ºC for 24h.
Day 2- 2018.02.13:
CONCENTRATION OF PLASMID ALCAM [ ]=0.3098 microg/microL.
*For every plate of 60mm of diameter –> 8 microg of DNA + 12 microL of Lipofectamine.
- ALCAM: 2 plates
- 4.96 microL of DNA in 500microL of Optimem.
- 24 microL of Lipo in 500mL of 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.
Day 3- 2018.02.07:
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Permeation with triton 0.3%:
- TWO CONDITIONS: PERMEATED-CELLS (Triton 0.3%) AND NON-PERMEATED CELLS (without this step)
- Wash with PBS 1x.
- Blocking solution -> 5% goat serum in PBS for 1h at RT.
- Remove the blocking solution and freeze at -80ºC.
Day 4 – 2018.02.08: Check the ALCAM transfection – ICC:
- Blocking solution: 5% goat serum in PBS
- Primary antibody: Mouse cmyc diluted 1/200
- Secondary antibody: GAM 488 + GAH594 IgG diluted 1/1000
- Vectashield mounting medium
Results: The samples were well transfected; althought a lot of cells were not transfected (maybe a problem in the time of the coating; because we leave the plates with the coating more hours). We are going to use the non-permeated cells.
