The cells are kept in plates of 100 mm (diameter) at a concentration of 1,5×106 cells each plate (with about 10 ml of medium).
The 21st cell passaging was done the second day of a HEK293 cell transfection in culture slides for CNTN1, NF140, NF155 and NF186:
- Remove the medium from the plates and wash with PBS 1x.
- Put 5 mL of trypsin in the plates and let them 1 minute in the incubator.
- Stop the reaction with HEK293 medium, and pass the whole solution to a 15 mL tube.
- Centrifuge 5 minutes at 1200 rpm.
- Remove the supernatant medium supernatant and resuspend the cells in 10 mL of new medium.
- Count the cells à put in a microtube 10 μl of the cell suspension and 10 μl of trypan blue, and perform the count with the machine (“Neuromuscular” protocol).
- Place the volume for 1,5×106 cells on each plate, and add the 10 mL of HEK293 medium.
