Day 1- 2018.02.14:
- Prepare 8 plates of 60mm of diameter with 24 coverslips of 9mm and 1 plate of 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.
- Manual counting: 6.35×10^6cells/mL
- For every plate of 60mm: 5ml of HEK medium + the volume that correspond to 800.000 of cells: 125microL.
- Incubate at 37ºC for 24h.
Day 2- 2018.02.15:
CONCENTRATION OF PLASMID ALCAM [ ]=0.3098 microg/microL.
CONCENTRATION OF PLASMID CONTACTIN []=1.181microg/microL.
*For every plate of 60mm of diameter –> 8 microg of DNA + 12 microL of Lipofectamine.
- Contactin : 5 plates
- 33.87 microL of DNA in 1.25 mL of Optimem.
- 60 microL of Lipo in 1.25mL of Optimem.
- ALCAM: 4 plates
- 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- 2018.02.16:
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Blocking solution:
- For CNTN1: Rabbit serum 1/40 in PBS for 1h at RT.
- For ALCAM: 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 and CNTN1 transfection – ICC:
For ALCAM:
- Blocking solution: 5% goat serum in PBS
- Primary antibody: Mouse cmyc diluted 1/200
- Secondary antibody: GAM 488 diluted 1/1000
- Vectashield mounting medium
For Contactin1:
- Blocking solution: Rabbit serum 1/40 in PBS
- Primary antibody: AF904 Diluted 1/1000
- Secondary antibody: RAG488 diluted 1/1000
- Vectashield mounting medium
Results: The samples were well transfected.
