Protocols for transfection of YCs expressed in cytosol
HeLa cells seeding:
- Seed 15.000 cells per dish (1,5 mL) in Mat-Tek
P35G-1.5-14-C dishes for 24 h.
Transfection (procedure for 1 dish):
- Prepare DNA solution: dilute 3 µL FuGENE in 100 µL Opti-MEM
and mix thoroughly, add 1 µg of DNA, mix again and incubate 30
min at room temperature.
- Wash the cells gently with PBS x 3.
- Add 1400 µL of Opti-MEM.
- Add the DNA solution prepared previously.
- Incubated at 37ºC overnight (8-10 h). Important:
Opti-MEM does not contain antibiotic!
- Remove transfection mixture.
- Wash gently with PBS x 3.
- Add DMEM high glucose + 10 % FCS.
- Allow cells to express YCs proteins for 3-6 days (incubate at 37ºC
and change medium as usual).
- Use medium without phenol red or the corresponding buffer for measurements.