Day 1- 2021.03.22:
- Prepare 12 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- 2021.03.23:
- 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] = 1,269 microg/microL
[NF140] = 1,07 microg/microL (maxiprep SIN TAG from glicerolate).
[NF155] =0,420 microg/microL.
[NF186]= 0,953 microg/microL.
- LIPOFECTAMINA: 96 wells
- 38,5 microL of Lipo in 820microL of Optimem
- CNTN1 : 24 wells
- 5,2 microL of DNA in 200 microL of Optimem.
- NF140: 24 wells
- 6,2 microL of DNA in 200 microL of Optimem.
- NF155: 24 wells
- 15,7 microL of DNA in 200 microL of Optimem.
- NF186: 24 wells
- 7 microL of DNA in 200 microL of Optimem.
Day 3- 2021.03.24:
- 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
