Day 1- 2021.03.10:
- Prepare 6 Corning® BioCoat™ 8-well culture slides à 354632 Corning.
- Coating with 0,3ml of Poly-D-Lysina (diluted 1/40 with sterile PBS x1)
- Incubate for 1h (37ºC, 5% CO2).
Day 2- 2021.03.11:
In each culture slide, we transfect 2 wells of each antigen (NF140, NF186, NF155, CNTN1). For 8 wells:
- 68 µL Optimem + 3,2 µL lipofectamina
- 68 µL Optimem + 2,2 µg DNA
- Incubate at RT for 5 min (in eppendorfs).
- Unify DNA with Lipofectamine and incubate at RT for 5min.
- In the meantime, seed the cells à For every well: 0,3 ml of HEK medium + the volume that correspond to 120.000 cells.
- Add 17 µL of the mix (DNA+lipo) to every well.
- Incubate O/N at 37ºC.
[CNTN1] =0,913 microg/microL
[NF140] =1.07microg/microL (maxiprep from glicerolate).
[NF155] =0,504microg/microL.
[NF186]=1,129microg/microL.
- LIPOFECTAMINA: 48 wells
- 19,2 microL of Lipo in 408microL of Optimem
- CNTN1 : 12 wells
- 3.6 microL of DNA in 120microL of Optimem.
- NF140: 12 wells
- 3.1 microL of DNA in 120microL of Optimem.
- NF155: 12 wells
- 6,6 microL of DNA in 120microL of Optimem.
- NF186: 12 wells
- 2.9microL of DNA in 120microL of Optimem.
Day 3- 2021.03.12:
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x and remove the chambers.
- Blocking solution: Goat serum 5% or Rabbit serum 1/40 in PBS for 1h at RT.
- Remove the blocking solution and freeze at -80ºC
