Objective
We need transfected HEKs grown in coverslips to test if positive sera for neurofascin make change them. We’ll do 2 35mm-plates with 5 coverslips, one for positive control and one for negative control.
Materials
- 35mm plate
- coverslips
- Poly-D-Lisine
- PBS
- HEK medium
- HEK cells
- pCMV6-NF155 (1ug/ul)
- Lipofectamine
- opti-MEM
Procedure
- Put 5 coverslips in a 35mm plate and incubate it at 37ºC with 2ml poly-D-lisine 1:500 for at least one hour.
- Wash 2 times with PBS and leave them dried
- Put HEK medium and 450.000 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
- Prepare primary antibody chicken-anti-neurofascin 1:2000 and incubate one coverslip per condition for 1h
- Wash 3 times with PBS
- Incubate coverslips 1h with secondary antibodies GAC488 + GAH594 1:500
Results & conclusions
Cells incubated o/n with sera with antibodies against NFASC were positive for Ab against human IgG and positive for NFASC, and they present adhesions.
Cells incubated o/n with sera without antibodies against NFASC only differ from the others in the marks of human IgG (in this case are negative).
