20150504 HEK transfections (CD9, gliomedin, NF186, NrCAM, NaVb1)

Objectives

To test if patients’ sera react against diferent protein present in de node of Ranvier.

Materials

• Coverslips
• 60mm cell culture plates
• Poly-D-lysine
• PBS
• HEK Medium (FBS, Horse serum, glutamine, pen-strep, pyruvate)
• Trypsine
• HEK293 cells
• Lipofectamine 2000
• Opti-MEM
• PcMV6-CD9-mycDDK
• CNTN-Human (Genecopoeia): EX-A1153-M02
• NrCAM: cDNA
• Gliomedin: cDNA
• Goat5%
• PFA4%
• Tritón 0.3%/PBS

Methods

04.05.2015

1. Put 12 coverslips per plate (x12)
2. Incubate 1h with poly-D-lysine 1/500 in PBS at 37ºC
3. Wash with PBS
4. Add HEK medium
5. Add 750000 cells per plate (250000 cells in 35mm plate)
6. Incubate it o/n at 37ºC

05.05.2015

1. Prepare DNA and lipofectamine (8ug of DNA in 250ul of OptiMEM and 12ul of lipofectamine in 250ml of OptiMEM per plate)
   1. CD9: 6ul DNA in 250ul OptiMEM + 12ul lipo in 250ul OptiMEM
   2. Gliomedin: 24ul DNA in 750ul OptiMEM + 36ul lipo in 750ul OptiMEM
   3. NF186: 10ul DNA in 500ul OptiMEM + 24ul lipo in 500ul OptiMEM
   4. NrCAM: 24ul DNA in 750ul OptiMEM + 36ul lipo in 750ul OptiMEM
   5. NaVb1: 59ul DNA in 750ul OptiMEM + 36ul lipo in 750ul OptiMEM
   6. CNTN-Huma (Genecopoeia): 3ugDNA/100OptiMEM + 4ul lipo/100 OptiMEM
2. Wait 5 minutes and mix the lipofectamine with the DNA
3. Incubate it 30 minutes and put 500ul of each solution per plate
4. Incubate the cells o/n at 37ºC
5. Wash cells with PBS and fis them with PFA 4% for 10 minutes
6. Wash with PBS
7. Permeabilization
   1. CNTN1-H + CD9: not permeabilized
   2. NF186 + NrCAM + Gliomedin + NaVb1: tritón 0.3% 5min + PBS wash
8. Blocking buffer: goat serum 5% at least 1hour.

Results 

An ICC is performed for NF186, CD9 and CNTN1H and the rest of the coverslips are frozen at -80ºC until needed.