Day 1- 2018.02.26:
- Prepare 4 plates of 60mm of diameter with 24 coverslips of 9mm.
- Treatment with Poly-D-Lysina (diluted 1/40 with PBS)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS 2x.
- Trypsin-EDTA a plate of HEK293.
- Manual counting: 5.6×10^6cells/mL
- For every plate of 60mm: 5ml of HEK medium + the volume that correspond to 1.000.000 of cells: 178.6microL.
- Incubate at 37ºC for 24h.
Day 2- 2018.02.27:
[ALCAM]=0.3098 microg/microL.
*For every plate of 60mm of diameter –> 8 microg of DNA + 12 microL of Lipofectamine.
- 103.29microL of DNA in 1mL of Optimem.
- 48 microL of Lipo in 1mL 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- 2018.02.28:
- 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
- Primary antibody: Mouse cmyc diluted 1/200
- Secondary antibody: GAM 488 diluted 1/1000
- Vectashield mounting medium
Results: The samples were well transfected.
