Day 1- 2018.04.09:
- Prepare 3 plates of 60mm of diameter with 24 coverslips (9 mm)
- Treatment with Poly-D-Lysine (diluted 1/40 with boratte buffer)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS 1x (4 times)
- Trypsin-EDTA a plate of HEK293.
- Counting
- For every plate: 5ml of HEK medium + the volume that correspond to 800.000 of cells
- Incubate at 37ºC for 24h.
Day 2- 2018.04.10:
[ANO2] = 590.1 ng/mcl
- For every plate –> 8 microg of DNA + 12 microL of Lipofectamine. In this case (x3 plates):
- 40,7 mcl of DNA in 0,75 ml of Optimem
- 36 mcl Lipo in 0,75 ml of Optimem.
- Incubate at RT for 5 min.
- Unify the DNA solution with the Lipo solution and incubate at RT for 30 min.
- For every plate: 500microL of the mix DNA-lipo
- Incubate O/N at 37ºC.
Day 3- 2018.04.11:
- Fix with PFA 4% (aprox 5 ml/plate). Incubation at RT during 10min.
- Wash with PBS 1x
- Permeabilize with Triton 0’3 % (only 2 of the 3 plates, the other one without permeabilization)
- 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:
- Take 2 coverslips from the plates with permeabilized cells, and 2 coverslips from the plates with non-permeabilized cells (blocked with goat serum 5%)
- Patient’s sera diluted 1/100 with 5 % Goat Serum. Samples:
- 151 (Multiple Sclerosis sera collection)
- Negative control
- Primary antibody: ANO2 (TA316534 OriGene) diluted 1/500
- Secondary antibody: GAR488 + GAH594 diluted 1/1000
- Vectashield mounting medium
Results: well transfected
