20150511 HEK transfection (NaVb2, L1CAM, CD9)

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

11.05.2015

1. Put 12 coverslips per plate (x9)
2. Incubate 1h with poly-D-lysine 1/500 in PBS at 37ºC
3. Wash with PBS
4. Add HEK medium
5. Add 700000 cells per plate
6. Incubate it o/n at 37ºC

12.05.2015

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. NaVb2:
   3. L1CAM:
   4. CNTN1-Human (Origene):
   5. NFASC-155-Human:
2. Wait 5 minutes and mix the lipofectamine with the DNA
3. Incubate it 30 minutes
4. During this incubation change the medium of the plates
5. Then, put 500ul of each transfection solution per plate
6. Incubate the cells o/n at 37ºC

13.05.2015

1. Wash cells with PBS and fix them with PFA 4% for 10 minutes
2. Wash with PBS
3. Permeabilization (CD9 + L1CAM): tritón 0.3% 5min + PBS wash
4. Wash three times with PBS
5. Blocking buffer: goat serum 5% at least 1hour.

Aspirate the blocking buffer and rapisly store the plates at -80ºC

Results 

Plates are stored at -80ºC.