20210316-18 – TRANSFECTION HEK293 IN CULTURE SLIDES FOR CNTN1, NF140, NF155, NF186

DAY 1 – 2021_03_16

• Prepare 6 Corning® BioCoat™ 8-well culture slides.
• Coating with 0,3ml of Poly-D-Lysina (diluted 1/40 with sterile PBS x1)
• Incubate overnight (37ºC, 5% CO2).

DAY 2 – 2021_03_17

• In each culture slide, we transfect 2 wells of each antigen (NF140, NF186, NF155, CNTN1). For this, the DNA and lipofectamine transfection solutions are prepared separately:
  • Reference volumes for 8 wells:
    • For DNA: 2,2 µg + 68 µL Optimem medium
    • For Lipofectamina: 3,2 µL + 68 µL Optimem medium
  • DNA concentrations:
    • [CNTN1] = 1,181 µg/µL
    • [NF140] = 1,07 µg/µL
    • [NF155] = 1,043 µg/µL
    • [NF186]= 0,953 µg/µL
• Volumes of transfection solutions for 48 wells (6 culture slides):
  • Lipofectamina (LF) (48 wells): 19,2 µL of LF in 408 µL of Optimem
  • CNTN1 (12 wells): 2,8 µL of DNA in 102 µL of Optimem.
  • NF140 (12 wells): 3,1 µL of DNA in 102 µL of Optimem.
  • NF155 (12 wells): 3,2 µL of DNA in 102 µL of Optimem.
  • NF186 (12 wells): 3,5 µL of DNA in 102 µL of Optimem.

• Incubate the solutions at room temperature (RT) for 5 min (in Eppendorf’s).
• Unify DNA with Lipofectamine and incubate at RT for 5min.
• In the meantime, seed the cells in each well: remove Poly-D-Lysina, add 0,3 ml of HEK medium and finally add the volume that correspond to 120.000 cells.
• Add 17 µL of the mix (DNA + lipofectamina) to every well.
• Incubate overnight at 37ºC.

DAY 3 – 2021_03_18

• Remove the mix of every well and add fix with PFA 4%. Incubation at RT fot 10min.
• Wash with PBS 1x and remove the chambers of the culture slides.
• Add blocking solution to each well: Goat serum 5% for NF140, NF155 and NF186 wells and Rabbit serum 1/40 for CTNTN1 wells for 1 hour at RT.
• Remove the blocking solution and freeze at -80ºC.