Objective
We need transfected HEK grown in coverslips to test patients sera against CNTN and NFASC. We’ll do 1 plate with 12 coverslips for CNTN1-human and 3 plates for NFASC.
Materials
- 60mm plate
- coverslips
- Poly-D-Lisine
- PBS
- HEK medium
- HEK cells
- pCMV6-CNTN human (733ng/ul)
- pCMV6-NF155 (1ug/ul)
- Lipofectamine
- opti-MEM
- HEK medium (FBS, DMEM, Horse serum, glutamine, piruvate, streptovidina-penicilina)
Procedure
- Put 10-12 coverslips in a 60mm plate and incubate it at 37ºC with 5ml poly-D-lisine 1:500 for at least one hour.
- Wash 2 times with PBS.
- Put HEK medium and 900.000 cels in each plate. Incubate it o/n
- Prepare DNA and lipofectamine with opti-MEM wait 5minutes and then put both together and wait 30minutes.
- Dismiss HEK medium from the plate
- Put new medium with lipofectamine and plasmid DNA. Incubate it o/n
- Aspirate off all medium
- Wash with PBS 2 times
- Fixation with PFA4% during 10 minutes
- Wash with PBS 3 times
- Blocking with PBS – Goat serum 5% 1h.
- Dismiss the Blocking and use coverslips or storage them at -80ºC
Results & conclusions
Some neurofascin trasnfected coverslips were used and the others were stored at -80ºC.
Contactin coverslips were used for ICC.
