Myoblasts electroporation

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Material

(Start) Lamina

  • Press bottom of ventilation
  • Switch on the light, put cable into the hood

(Finish) Lamina

  • Press ventilation button until light is of
  • Switch down the lid and switch on UV-light

other Materials

  • HS AMEM (25°C)
  • APBS (100ml PBS and 25ml H2O)
  • 50ml tube of 0.75% gelatin (stored @ 4°C and prewarm @ 37°C)
  • 0.05% Trypsin/EDTA (36ml APBS+4ml 10x TE) (stored @ 4°C and prewarm @ 25°C)

Procedure

  1. Coat the 4-well plates with gelatine for 5 min before aspirate
  2. Aspirate the medium off of the flast containing the cells
  3. Rinse the flask with 7ml APBS (5ml is also enough)
  4. Wash the cells: aspirate off the APBS
  5. Add 5ml TE to the flask, shake it gently and check under the microscope
  6. Stop reaction with HS MEM (7ml)
  7. Mix well with a pipette also over the cells
  8. Put the cells into a 15ml tube and centrifuge for 3' @ 1000rpm
  9. Prepare the tubes for electroporation: add 5µl in each chamber
  10. Aspirate liquid from the cells and resuspend the cells in 50µl buffer R (Steinberg) (120-140µl)
  11. Add 12µl of resuspended cells into each tube (additionally to the electroporated plasmids) into 1x Steinberg
  12. Electroporator (PEQLAB):
    1. Take the device under the hood
    2. Fill the solution into the chamber (add 3ml electrolyte solution (red label: E) into a special plastic container
    3. Settings: 800V, pulse nr. 3, 35ms pulse length
  13. Add electroporated cells to the prepared media (300µl each tube)
  14. Add cells into the chambers of a 4x chamber slide
  15. Incubate cells @ 25°C
  16. Change the medium after 24h
  17. Fixation
    1. Take out the cells and suck off the medium
    2. Wash 3x30s with APBS
    3. Add 1x MEMFA to the cells and fix for 20‘ @ RT
    4. Proceed with the antibody-staining as usual