Electroporation of DAPI into Dictyostelium

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Procedure

1. Give fresh HL5 medium to all plates to be resuspended.


2. Resuspend wildtype cells from 2 or 3 plates and transfer to a sterile 50 ml conical tube.


3. Pellet cells by spinning in tabletop centrifuge at 2,000 rpm for 5 minutes.


4. Draw off HL5 medium and replace with COLD H50 buffer. Be sure to not disturb the cell pellet. Resuspend cells to wash and pellet as before.


5. Draw off 1st H50 wash and replace with a 2nd 10ml aliquot. Resuspend cells to wash again and remove a small aliquot using a Pasteur pipette and bulb. Add a drop of resuspension to each side of grid on hemacytometer. Gently place cover glass over grid.


6. Note remaining volume of resuspension and pellet as before.


7. Count cells on grid and determine the total number of cells in tube.


8. Draw off 2nd H50 wash and replace with an appropriate volume of H50 such that the final cell density is 5 x 10⁷ cells/ml.


9. Thaw DAPI in DMSO stock solution. Dilute DAPI in 100 µl cell/H50 suspension such that final DAPI concentration is 1.5 µg/ml. Cell suspension and DAPI are mixed in a 0.5 ml microfuge tube. Use DAPI stock solution at a concentration such that the amount of DMSO added to reaction is minimized (use highest concentration of DAPI stock solution possible).


10. Place cells/DAPI in a pre-chilled 0.1 mm cuvette; leave cuvette on ice for 3 minutes.


11. Place cuvette in cold cuvette holder. Zap (pulse or electroporate) cells @ 0.85 kV and 25 µF by depressing two red buttons simultaneously until a beep is heard. Record time constants.


12. Immediately following electroporation, add 1 ml of HL5 medium to cuvette. Leave cuvette at room temperature for 10 minutes.


13. Use gel loading pipette tip to remove ~200 µl aliquots from cuvette (don't forget to remove cells between electrodes) and add to a coated coverslip.


14. Allow cells to settle onto coverslip for 1hr before adding an additional 1 ml HL5 medium to well. Add medium to edge of well such that cells attached to coverslip are not disturbed. Allow cells to recover for additional 3 hrs.


15. Observe cells on an inverted scope using oil immersion lens and DAPI filter. Neutral density filters may be needed to minimize glare caused by intense staining.

Materials

• H-50 buffer
  
  
  • 20mM Hepes
  • 50mM KCl
  • 10mM NaCl
  • 1mM Mg₂SO₄
  • 5 mMNaHCO₃
  • 1mM Na₂HPO₄
  
  Adjust pH to 7.0

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